Cytotoxicity mediation of cells evidencing surface expression of TROP-2

ABSTRACT

The present invention relates to a method for producing cancerous disease modifying antibodies using a novel paradigm of screening. By segregating the anti-cancer antibodies using cancer cell cytotoxicity as an end point, the process makes possible the production of anti-cancer antibodies for therapeutic and diagnostic purposes. The antibodies can be used in aid of staging and diagnosis of a cancer, and can be used to treat primary tumors and tumor metastases. The anti-cancer antibodies can be conjugated to toxins, enzymes, radioactive compounds, cytokines, interferons, target or reporter moieties and hematogenous cells.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/709,898, filed on Feb. 22, 2007, which claims benefit of the filingdate of U.S. Provisional Patent Application No. 60/776,466, filed onFeb. 24, 2006, the contents of which are herein incorporated byreference.

FIELD OF THE INVENTION

This invention relates to the isolation and production of cancerousdisease modifying antibodies (CDMAB) and to the use of these CDMAB intherapeutic and diagnostic processes, optionally in combination with oneor more chemotherapeutic agents. The invention further relates tobinding assays which utilize the CDMAB of the instant invention.

BACKGROUND OF THE INVENTION

TROP-2 is a cell surface glycoprotein expressed on most carcinomas, aswell as some normal human tissues. It was initially defined as amolecule recognized by two murine monoclonal antibodies raised to ahuman choriocarcinoma cell line BeWo that recognized an antigen on humantrophoblast cells (Faulk 1978). The same molecule was independentlydiscovered by other investigators which led to multiple names describingthe same antigen. Hence, TROP-2 was also referred to as GA733-1 andepithelial glycoprotein-1 (EGP-1) (Basu 1995, Formaro 1995).

The TROP-2 gene is an intronless gene that was thought to have beenformed through the retroposition of a homologous gene GA 733-2 (alsoknown as epithelial glycoprotein-2, EpCAM and Trop-1) via an RNAintermediate. The TROP-2 gene has been mapped to chromosome 1p32(Calabrese 2001). The protein component of TROP-2 has a molecular massof approximately 35 kilodaltons. Its mass may be increased by 11-13kilodaltons with heterogeneous N-linked glycosylation of itsextracellular domain. There are many cysteine residues in theextracellular domain which could form disulfide bridge sites. TROP-2 isa substrate for protein kinase C, a Ca²⁺ dependent protein kinase andthe intracellular serine 303 residue has been shown to be phosphorylated(Basu 1995). It has also been shown that crossing-linking of TROP-2 withanti-TROP-2 antibodies transduced a calcium signal as shown by a rise incytoplasmic Ca²⁺ (Ripani 1998). These data support signal transductionas a physiological function of TROP-2, although to date no physiologicalligand has been identified. Recently an association between TROP-2expression and cancer has been shown as TROP-2 was identified as amember of a group of genes reported to be the most highly overexpressedin ovarian serous papillary carcinoma compared to normal ovarianepithelium in a large-scale gene expression analysis using cDNAmicroarray technology (Santin 2004).

The expression profile of TROP-2 has been elucidated throughimmunohistochemistry (IHC) and flow cytometery studies using manydifferent TROP-2 antibodies. Anti-TROP-2 antibodies 162-25.3 and162-46.2 were produced through immunization of mice with the humanchoriocarcinoma cell line BeWo, and were investigated for theirreactivity to a series of tumor and lymphoid cell lines and peripheralblood mononuclear cells. In this study both antibodies appeared to betrophoblast specific, staining 3 of the 4 choriocarcinoma cell linestested, while none of the other lymphoid or tumor cell lines(representing fibrosarcoma, cervical sarcoma, colon carcinoma, melanoma,neuroblastoma, erythroleukemia) were stained in an indirectimmunofluorescence FACS assay. In addition, none of the normalperipheral blood cells were stained. The antibodies were tested forstaining of formalin-fixed paraffin-embedded placenta tissue sectionsand frozen normal sections of liver, kidney, spleen, thymus and lymphnode tissues. The placenta tissue sections were stained with bothantibodies, while there was no staining of the other normal tissues(Lipinski 1981). These two antibodies have strictly been reported foruse in in vitro diagnostic studies.

Anti-TROP-2 antibody MOv16 was generated through the immunization ofmice with a crude membrane preparation of poorly differentiated ovariancarcinoma OvCa4343/83. MOv16 was tested for reactivity to a series offrozen tissue sections of benign and malignant ovarian tumors. MOv16reacted with 31 of 54 malignant ovarian tumors and 2 of 16 benignovarian tumors. Of the 5 mucinous ovarian tumors that were tested, MOv16was completely unreactive. MOv16 was also tested for reactivity tofrozen sections of non-ovarian malignant tumors where it was found tobind 117 of 189 breast carcinoma sections and 12 of 18 lung carcinomasections. MOv16 was completely unreactive on 16 non-epithelial tumorsthat were tested (including liposarcomas, chondrosarcomas,endotheliomas, histiocytomas and dysgerminomas). When tested on frozennormal tissue sections, MOv-16 was reactive with breast, pancreas,kidney and prostate sections. MOv16 reactivity was reported to benegative on lung, spleen, skin, ovary, thyroid, parotid gland, stomach,larynx, uterus and colon sections, though the number of tissue sectionsthat were used was not reported. The authors noted that frozen tissuesections were used because MOv16 was unreactive to paraffin embeddedtissues (Miotti 1987). This antibody has also only been reported for usein in vitro diagnostic studies.

Anti-TROP-2 antibody Rs7-3G11 (RS7) was generated through theimmunization of mice with a crude membrane preparation derived from asurgically removed human primary squamous cell carcinoma of the lung.IHC was used to examine the staining of RS7 on frozen sections of humantumor and normal tissues. RS7 bound to 33 of the 40 sectionsrepresenting tumors of the breast, colon, kidney, lung, prostate andsquamous cell cancer. Of the normal tissues RS7 bound to 16 of 20sections of breast, colon, kidney, liver, lung and prostate tissueswhile none of the five sections of spleen tissue were stained. In thisstudy the authors noted that it appeared that antigen density in tumorswas higher than in normal epithelial tissues (Stein 1990).

Additional studies of the tissue specificity of RS7 were carried out onboth tumor and normal tissues. RS7 was tested on a panel of frozen tumorsections and bound to 65 of the 77 sections representing tumors of thelung, stomach, kidney, bladder, colon, breast, ovary, uterus andprostate. There was no binding to the 5 lymphomas tested. RS7 was testedon a panel of 85 frozen human normal tissue sections composed of a totalof 24 tissue types. 39 sections of 13 normal tissues (lung, bronchus,trachea, esophagus, colon, liver, pancreas, kidney, bladder, skin,thyroid, breast and prostate) were stained by RS7. The authors of thisstudy noted that in the tissues in which positive staining was observed,the reactivity was generally restricted to epithelial cells, primarilyin ducts or glands. It was also noted that this study was limited tofrozen sections since it was observed that RS7 was not reactive onformalin-fixed paraffin-embedded sections (Stein 1993).

Polyclonal anti-TROP-2 antibodies were prepared by immunizing mice witha synthetic peptide corresponding to amino acid positions between 169and 182 of the cytoplasmic domain of human TROP-2. The polyclonalantibodies were tested on a tissue array slide that containedformalin-fixed human esophageal hyperplasia and carcinoma tissues. Tenof the 55 carcinoma specimens displayed heavy staining with thepolyclonal antibodies, while the mild hyperplasia tissue stained veryweakly, indicating expression levels may be related to malignanttransformation (Nakashima 2004).

Overall, IHC reactivity patterns obtained with different anti-TROP-2antibodies were consistent. Expression in cancer was seen primarily incarcinomas, and most carcinomas were reactive. In normal tissues,expression appeared to be limited to cells of epithelial origin, andthere was some evidence that staining of carcinomas was stronger thanstaining of corresponding normal epithelial tissues.

In addition to being used in IHC studies, antibody RS7 was tested in invivo models with initial experiments consisting of tumor targetingstudies in nude mouse xenograft models. Radiolabeled RS7 injected i.v.was shown to accumulate specifically in the tumor of mice bearing eitherCalu-3 (lung adenocarcinoma) or GW-39 (colon carcinoma) tumors (Stein1990). Further studies were done to investigate the biodistribution ofradiolabeled RS7 in a xenograft system and to study the therapeuticpotential of RS7 as an immunoconjugate. In this study the therapeuticefficacy of ¹³¹I-labeled RS7 F(ab′)₂ was investigated in nude micebearing Calu-3 human lung adenocarcinoma xenografts. Three weeksfollowing inoculation of the mice with Calu-3 cells, when the tumors hadreached a size of approximately 0.3-0.9 grams, groups of 6-7 mice weretreated with a single dose i.v. of either 10.0 mCi ¹³¹I-RS7-F(ab′)₂ or1.5 mCi 131I-RS7-F(ab′)₂ and compared to a similar group of untreatedcontrol mice. The single dose of 1.0 mCi ¹³¹I-RS7-F(ab′)₂ resulted intumor growth suppression for approximately 5 weeks, while the singledose of 1.5 mCi ¹³¹I-RS7-F(ab′)₂ resulted in tumor regression, and themean tumor size did not exceed the pre-therapy size until the eighthweek after radioantibody injection. Mice receiving the 1.5 mCi¹³¹I-RS7-F(ab′)₂ dose experienced a mean body weight loss of 18.7percent, indicating there was toxicity associated with the treatment. Inthis study, effects of treatment with naked RS7 or the F(ab′)₂ fragmentof RS7 were not tested (Stein 1994a). Another study was done to test theefficacy of ¹³¹I-RS7 in a MDA-MB-468 breast cancer xenograft model.Groups of ten mice bearing MDA-MB-468 tumors of approximately 0.1 cm³were treated with a single dose i.v. of either 250 microcuries ¹³¹I-RS7or 250 microcuries ¹³¹I-Ag8 (an isotype matched control antibody).Groups of six mice were treated with a single dose i.v. of 30 microgramsof either unlabeled RS7 or Ag8. Complete regression of the tumors(except for one animal that had a transient reappearance of tumor) wasseen in the animals treated with ¹³¹I-RS7, which lasted for the durationof the 11 week observation period. Tumor regression was also seen in¹³¹I-Ag8 treated mice, though was only observed between 2 weeks and 5weeks with tumors either persisting or continuing to grow for theremainder of the study. Tumor growth of mice that received unlabeled RS7or Ag8 was not inhibited and there did not appear to be any differencesin the mean tumor volume of RS7 treated mice compared to the Ag8 treatedmice. Two additional groups of 10 mice bearing larger MDA-MB-468 tumorsof approximately 0.2-0.3 cm³ were treated with a slightly higher singledose of either 275 microcuries ¹³¹I-Rs7 or 275 microcuries ¹³¹Ag8 andcompared to a similar group of untreated mice. Tumor volume was measuredweekly for 15 weeks. Although in this case there was a significantdifference in tumor growth between the ¹³¹I-RS7 treated mice compared tothe untreated mice, there was no significant difference in the tumorgrowth of the ¹³¹I-RS7 compared to the ¹³¹I-Ag8 treated mice, indicatinga portion of the efficacy may have been due to non-specific effects ofthe radiation. Unlabeled antibodies were not tested in mice containing0.2-0.3 cm³ tumors (Shih 1995).

There have been numerous additional studies examining the efficacy ofRS7 as an immunoconjugate with an attempt to select the optimalradiolabel for radioimmunotherapy (Stein 2001a, Stein 2001b, Stein2003). A humanized version of RS7 has also been generated, however ithas only been tested in preclinical xenograft models as a radioconjugate(Govindan 2004). These studies show similar positive effects as thepreviously described studies with RS7, however in one study, even whenradiolabeled RS7 was delivered at a previously determined maximumtolerable dose, toxicity occurred leading to death in some mice (Stein2001a). Although effective treatment of xenograft tumors in mice wasachieved with radiolabeled RS7 in these studies, naked RS7 was notevaluated.

Immunizing mice with neuramindase pre-treated H3922 human breastcarcinoma cells produced the anti-TROP-2 monoclonal antibody BR110 (asdisclosed in U.S. Pat. No. 5,850,854, refer to Prior Patents section).By immunohistology, using human frozen tissue specimens, BR110 was shownto react with a wide range of human carcinoma specimens including thoseof the lung, colon, breast, ovarian, kidney, esophagus, pancreas, skin,lung and tonsil. No human normal tissue sections were tested. In vitrostudies demonstrated that BR110 had no ADCC or CDC activity on the humancarcinoma cell lines H3396 or H3922. In vitro studies analyzing thecytotoxicity of BR110-immunotoxins was performed on the human cancercell lines H3619, H2987, MCF-7, H3396 and H2981. The EC₅₀ for the celllines tested was 0.06, 0.001, 0.05, 0.09 and >5 micrograms/mLrespectively. No cytotoxicity data was disclosed for the naked BR110antibody. No in vivo data was disclosed for the naked orimmunoconjugated BR110.

A number of additional antibodies have been generated that targetTROP-2, such as MR54, MR6 and MR23 which were generated fromimmunization of mice with the ovarian cancer cell line Colo 316 (Stein1994b) and antibody T16 which was generated by immunization of mice withthe T24 bladder cancer cell line (Fradet 1984). The use of theseantibodies has been limited to biochemical characterization of theTROP-2 antigen and cell line and tissue expression studies. There havebeen no reports of anti-cancer efficacy of these antibodies, either invitro or in vivo. RS7 was the only antibody that was tested fortherapeutic efficacy in preclinical cancer models, with its use beinglimited to a carrier of radioisotope. There are no reports of any nakedTROP-2 antibodies exhibiting therapeutic efficacy in preclinical cancermodels either in vitro or in vivo.

Monoclonal Antibodies as Cancer Therapy: Each individual who presentswith cancer is unique and has a cancer that is as different from othercancers as that person's identity. Despite this, current therapy treatsall patients with the same type of cancer, at the same stage, in thesame way. At least 30 percent of these patients will fail the first linetherapy, thus leading to further rounds of treatment and the increasedprobability of treatment failure, metastases, and ultimately, death. Asuperior approach to treatment would be the customization of therapy forthe particular individual. The only current therapy which lends itselfto customization is surgery. Chemotherapy and radiation treatment cannotbe tailored to the patient, and surgery by itself, in most cases isinadequate for producing cures.

With the advent of monoclonal antibodies, the possibility of developingmethods for customized therapy became more realistic since each antibodycan be directed to a single epitope. Furthermore, it is possible toproduce a combination of antibodies that are directed to theconstellation of epitopes that uniquely define a particular individual'stumor.

Having recognized that a significant difference between cancerous andnormal cells is that cancerous cells contain antigens that are specificto transformed cells, the scientific community has long held thatmonoclonal antibodies can be designed to specifically target transformedcells by binding specifically to these cancer antigens; thus giving riseto the belief that monoclonal antibodies can serve as “Magic Bullets” toeliminate cancer cells. However, it is now widely recognized that nosingle monoclonal antibody can serve in all instances of cancer, andthat monoclonal antibodies can be deployed, as a class, as targetedcancer treatments. Monoclonal antibodies isolated in accordance with theteachings of the instantly disclosed invention have been shown to modifythe cancerous disease process in a manner which is beneficial to thepatient, for example by reducing the tumor burden, and will variously bereferred to herein as cancerous disease modifying antibodies (CDMAB) or“anti-cancer” antibodies.

At the present time, the cancer patient usually has few options oftreatment. The regimented approach to cancer therapy has producedimprovements in global survival and morbidity rates. However, to theparticular individual, these improved statistics do not necessarilycorrelate with an improvement in their personal situation.

Thus, if a methodology was put forth which enabled the practitioner totreat each tumor independently of other patients in the same cohort,this would permit the unique approach of tailoring therapy to just thatone person. Such a course of therapy would, ideally, increase the rateof cures, and produce better outcomes, thereby satisfying a long-feltneed.

Historically, the use of polyclonal antibodies has been used withlimited success in the treatment of human cancers. Lymphomas andleukemias have been treated with human plasma, but there were fewprolonged remission or responses. Furthermore, there was a lack ofreproducibility and there was no additional benefit compared tochemotherapy. Solid tumors such as breast cancers, melanomas and renalcell carcinomas have also been treated with human blood, chimpanzeeserum, human plasma and horse serum with correspondingly unpredictableand ineffective results.

There have been many clinical trials of monoclonal antibodies for solidtumors. In the 1980s there were at least four clinical trials for humanbreast cancer which produced only one responder from at least 47patients using antibodies against specific antigens or based on tissueselectivity. It was not until 1998 that there was a successful clinicaltrial using a humanized anti-Her2/neu antibody (Herceptin®) incombination with CISPLATIN. In this trial 37 patients were assessed forresponses of which about a quarter had a partial response rate and anadditional quarter had minor or stable disease progression. The mediantime to progression among the responders was 8.4 months with medianresponse duration of 5.3 months.

Herceptin® was approved in 1998 for first line use in combination withTaxol®. Clinical study results showed an increase in the median time todisease progression for those who received antibody therapy plus Taxol®(6.9 months) in comparison to the group that received Taxol® alone (3.0months). There was also a slight increase in median survival; 22 versus18 months for the Herceptin® plus Taxol® treatment arm versus the Taxol®treatment alone arm. In addition, there was an increase in the number ofboth complete (8 versus 2 percent) and partial responders (34 versus 15percent) in the antibody plus Taxol® combination group in comparison toTaxol® alone. However, treatment with Herceptin® and Taxol® led to ahigher incidence of cardiotoxicity in comparison to Taxol® treatmentalone (13 versus 1 percent respectively). Also, Herceptin® therapy wasonly effective for patients who over express (as determined throughimmunohistochemistry (1HC) analysis) the human epidermal growth factorreceptor 2 (Her2/neu), a receptor, which currently has no known functionor biologically important ligand; approximately 25 percent of patientswho have metastatic breast cancer. Therefore, there is still a largeunmet need for patients with breast cancer. Even those who can benefitfrom Herceptin® treatment would still require chemotherapy andconsequently would still have to deal with, at least to some degree, theside effects of this kind of treatment.

The clinical trials investigating colorectal cancer involve antibodiesagainst both glycoprotein and glycolipid targets. Antibodies such as17-1A, which has some specificity for adenocarcinomas, has undergonePhase 2 clinical trials in over 60 patients with only 1 patient having apartial response. In other trials, use of 177-1A produced only 1complete response and 2 minor responses among 52 patients in protocolsusing additional cyclophosphamide. To date, Phase III clinical trials of17-1A have not demonstrated improved efficacy as adjuvant therapy forstage III colon cancer. The use of a humanized murine monoclonalantibody initially approved for imaging also did not produce tumorregression.

Only recently have there been any positive results from colorectalcancer clinical studies with the use of monoclonal antibodies. In 2004,ERBITUX® was approved for the second line treatment of patients withEGFR-expressing metastatic colorectal cancer who are refractory toirinotecan-based chemotherapy. Results from both a two-arm Phase IIclinical study and a single arm study showed that ERBITUX® incombination with irinotecan had a response rate of 23 and 15 percentrespectively with a median time to disease progression of 4.1 and 6.5months respectively. Results from the same two-arm Phase II clinicalstudy and another single arm study showed that treatment with ERBITUX®alone resulted in an 11 and 9 percent response rate respectively with amedian time to disease progression of 1.5 and 4.2 months respectively.

Consequently in both Switzerland and the United States, ERBITUX®treatment in combination with irinotecan, and in the United States,ERBITUX® treatment alone, has been approved as a second line treatmentof colon cancer patients who have failed first line irinotecan therapy.Therefore, like Herceptin®, treatment in Switzerland is only approved asa combination of monoclonal antibody and chemotherapy. In addition,treatment in both Switzerland and the US is only approved for patientsas a second line therapy. Also, in 2004, AVASTIN® was approved for usein combination with intravenous 5-fluorouracil-based chemotherapy as afirst line treatment of metastatic colorectal cancer. Phase III clinicalstudy results demonstrated a prolongation in the median survival ofpatients treated with AVASTIN® plus 5-fluorouracil compared to patientstreated with 5-fluourouracil alone (20 months versus 16 monthsrespectively). However, again like Herceptin® and ERBITUX®, treatment isonly approved as a combination of monoclonal antibody and chemotherapy.

There also continues to be poor results for lung, brain, ovarian,pancreatic, prostate, and stomach cancer. The most promising recentresults for non-small cell lung cancer came from a Phase II clinicaltrial where treatment involved a monoclonal antibody (SGN-15; dox-BR96,anti-Sialyl-LeX) conjugated to the cell-killing drug doxorubicin incombination with the chemotherapeutic agent TAXOTERE®. TAXOTERE® is theonly FDA approved chemotherapy for the second line treatment of lungcancer. Initial data indicate an improved overall survival compared toTAXOTERE® alone. Out of the 62 patients who were recruited for thestudy, two-thirds received SGN-15 in combination with TAXOTERE® whilethe remaining one-third received TAXOTERE® alone. For the patientsreceiving SGN-15 in combination with TAXOTERE®, median overall survivalwas 7.3 months in comparison to 5.9 months for patients receivingTAXOTERE® alone. Overall survival at 1 year and 18 months was 29 and 18percent respectively for patients receiving SNG-15 plus TAXOTERE®compared to 24 and 8 percent respectively for patients receivingTAXOTERE® alone. Further clinical trials are planned.

Preclinically, there has been some limited success in the use ofmonoclonal antibodies for melanoma. Very few of these antibodies havereached clinical trials and to date none have been approved ordemonstrated favorable results in Phase III clinical trials.

The discovery of new drugs to treat disease is hindered by the lack ofidentification of relevant targets among the products of 30,000 knowngenes that could contribute to disease pathogenesis. In oncologyresearch, potential drug targets are often selected simply due to thefact that they are over-expressed in tumor cells. Targets thusidentified are then screened for interaction with a multitude ofcompounds. In the case of potential antibody therapies, these candidatecompounds are usually derived from traditional methods of monoclonalantibody generation according to the fundamental principles laid down byKohler and Milstein (1975, Nature, 256, 495-497, Kohler and Milstein).Spleen cells are collected from mice immunized with antigen (e.g. wholecells, cell fractions, purified antigen) and fused with immortalizedhybridoma partners. The resulting hybridomas are screened and selectedfor secretion of antibodies which bind most avidly to the target. Manytherapeutic and diagnostic antibodies directed against cancer cells,including Herceptin® and RITUXIMAB, have been produced using thesemethods and selected on the basis of their affinity. The flaws in thisstrategy are two-fold. Firstly, the choice of appropriate targets fortherapeutic or diagnostic antibody binding is limited by the paucity ofknowledge surrounding tissue specific carcinogenic processes and theresulting simplistic methods, such as selection by overexpression, bywhich these targets are identified. Secondly, the assumption that thedrug molecule that binds to the receptor with the greatest affinityusually has the highest probability for initiating or inhibiting asignal may not always be the case.

Despite some progress with the treatment of breast and colon cancer, theidentification and development of efficacious antibody therapies, eitheras single agents or co-treatments, have been inadequate for all types ofcancer.

Prior Patents:

U.S. Pat. No. 5,750,102 discloses a process wherein cells from apatient's tumor are transfected with MHC genes which may be cloned fromcells or tissue from the patient. These transfected cells are then usedto vaccinate the patient.

U.S. Pat. No. 4,861,581 discloses a process comprising the steps ofobtaining monoclonal antibodies that are specific to an internalcellular component of neoplastic and normal cells of the mammal but notto external components, labeling the monoclonal antibody, contacting thelabeled antibody with tissue of a mammal that has received therapy tokill neoplastic cells, and determining the effectiveness of therapy bymeasuring the binding of the labeled antibody to the internal cellularcomponent of the degenerating neoplastic cells. In preparing antibodiesdirected to human intracellular antigens, the patentee recognizes thatmalignant cells represent a convenient source of such antigens.

U.S. Pat. No. 5,171,665 provides a novel antibody and method for itsproduction. Specifically, the patent teaches formation of a monoclonalantibody which has the property of binding strongly to a protein antigenassociated with human tumors, e.g. those of the colon and lung, whilebinding to normal cells to a much lesser degree.

U.S. Pat. No. 5,484,596 provides a method of cancer therapy comprisingsurgically removing tumor tissue from a human cancer patient, treatingthe tumor tissue to obtain tumor cells, irradiating the tumor cells tobe viable but non-tumorigenic, and using these cells to prepare avaccine for the patient capable of inhibiting recurrence of the primarytumor while simultaneously inhibiting metastases. The patent teaches thedevelopment of monoclonal antibodies which are reactive with surfaceantigens of tumor cells. As set forth at col. 4, lines 45 et seq., thepatentees utilize autochthonous tumor cells in the development ofmonoclonal antibodies expressing active specific immunotherapy in humanneoplasia.

U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen characteristic ofhuman carcinomas and not dependent upon the epithelial tissue of origin.

U.S. Pat. No. 5,783,186 is drawn to Anti-Her2 antibodies which induceapoptosis in Her2 expressing cells, hybridoma cell lines producing theantibodies, methods of treating cancer using the antibodies andpharmaceutical compositions including said antibodies.

U.S. Pat. No. 5,849,876 describes new hybridoma cell lines for theproduction of monoclonal antibodies to mucin antigens purified fromtumor and non-tumor tissue sources.

U.S. Pat. No. 5,869,268 is drawn to a method for generating a humanlymphocyte producing an antibody specific to a desired antigen, a methodfor producing a monoclonal antibody, as well as monoclonal antibodiesproduced by the method. The patent is particularly drawn to theproduction of an anti-HD human monoclonal antibody useful for thediagnosis and treatment of cancers.

U.S. Pat. No. 5,869,045 relates to antibodies, antibody fragments,antibody conjugates and single-chain immunotoxins reactive with humancarcinoma cells. The mechanism by which these antibodies function istwo-fold, in that the molecules are reactive with cell membrane antigenspresent on the surface of human carcinomas, and further in that theantibodies have the ability to internalize within the carcinoma cells,subsequent to binding, making them especially useful for formingantibody-drug and antibody-toxin conjugates. In their unmodified formthe antibodies also manifest cytotoxic properties at specificconcentrations.

U.S. Pat. No. 5,780,033 discloses the use of autoantibodies for tumortherapy and prophylaxis. However, this antibody is an antinuclearautoantibody from an aged mammal. In this case, the autoantibody is saidto be one type of natural antibody found in the immune system. Becausethe autoantibody comes from “an aged mammal”, there is no requirementthat the autoantibody actually comes from the patient being treated. Inaddition the patent discloses natural and monoclonal antinuclearautoantibody from an aged mammal, and a hybridoma cell line producing amonoclonal antinuclear autoantibody.

U.S. Pat. No. 5,850,854 discloses a specific antibody, BR110 directedagainst GA733-1. This patent discloses in vitro function for BR110 as animmunotoxin conjugate. There was no in vitro function as a nakedantibody disclosed for this antibody. There was also no in vivo functiondisclosed for this antibody.

U.S. Pat. No. 6,653,104 claims immunotoxin-conjugated antibodies,including but not limited to RS7, directed against a host of antigens,including but not limited to EGP-1. The immunotoxin is limited to thosepossessing ribonucleolytic activity. However, the examples disclose onlya specific immunotoxin-conjugated antibody, LL2, directed against CD22.There was no in vitro or in vivo function for RS7 disclosed in thisapplication.

U.S. Application No. 20040001825A1 discloses a specific antibody, RS7directed against EGP-1. This application discloses in vitro function forRS7 as a radiolabeled conjugate. There was no in vitro function as anaked antibody disclosed for this antibody. This application alsodiscloses in vivo function for RS7 resulting from radiolabeled andunlabeled conjugate administered sequentially. However, this study waslimited to one patient and it is unknown whether any of the observedfunction was due to the unlabeled antibody. There was no in vivofunction for RS7 resulting from the administration of the nakedantibody.

SUMMARY OF THE INVENTION

This application utilizes methodology for producing patient specificanti-cancer antibodies taught in the U.S. Pat. No. 6,180,357 patent forisolating hybridoma cell lines which encode for cancerous diseasemodifying monoclonal antibodies. These antibodies can be madespecifically for one tumor and thus make possible the customization ofcancer therapy. Within the context of this application, anti-cancerantibodies having either cell-killing (cytotoxic) or cell-growthinhibiting (cytostatic) properties will hereafter be referred to ascytotoxic. These antibodies can be used in aid of staging and diagnosisof a cancer, and can be used to treat tumor metastases. These antibodiescan also be used for the prevention of cancer by way of prophylactictreatment. Unlike antibodies generated according to traditional drugdiscovery paradigms, antibodies generated in this way may targetmolecules and pathways not previously shown to be integral to the growthand/or survival of malignant tissue. Furthermore, the binding affinitiesof these antibodies are suited to requirements for initiation of thecytotoxic events that may not be amenable to stronger affinityinteractions. Also, it is within the purview of this invention toconjugate standard chemotherapeutic modalities, e.g. radionuclides, withthe CDMAB of the instant invention, thereby focusing the use of saidchemotherapeutics. The CDMAB can also be conjugated to toxins, cytotoxicmoieties, enzymes e.g. biotin conjugated enzymes, cytokines,interferons, target or reporter moieties or hematogenous cells, therebyforming an antibody conjugate. The CDMAB can be used alone or incombination with one or more CDMAB/chemotherapeutic agents.

The prospect of individualized anti-cancer treatment will bring about achange in the way a patient is managed. A likely clinical scenario isthat a tumor sample is obtained at the time of presentation, and banked.From this sample, the tumor can be typed from a panel of pre-existingcancerous disease modifying antibodies. The patient will beconventionally staged but the available antibodies can be of use infurther staging the patient. The patient can be treated immediately withthe existing antibodies, and a panel of antibodies specific to the tumorcan be produced either using the methods outlined herein or through theuse of phage display libraries in conjunction with the screening methodsherein disclosed. All the antibodies generated will be added to thelibrary of anti-cancer antibodies since there is a possibility thatother tumors can bear some of the same epitopes as the one that is beingtreated. The antibodies produced according to this method may be usefulto treat cancerous disease in any number of patients who have cancersthat bind to these antibodies.

In addition to anti-cancer antibodies, the patient can elect to receivethe currently recommended therapies as part of a multi-modal regimen oftreatment. The fact that the antibodies isolated via the presentmethodology are relatively non-toxic to non-cancerous cells allows forcombinations of antibodies at high doses to be used, either alone, or inconjunction with conventional therapy. The high therapeutic index willalso permit re-treatment on a short time scale that should decrease thelikelihood of emergence of treatment resistant cells.

If the patient is refractory to the initial course of therapy ormetastases develop, the process of generating specific antibodies to thetumor can be repeated for re-treatment. Furthermore, the anti-cancerantibodies can be conjugated to red blood cells obtained from thatpatient and re-infused for treatment of metastases. There have been feweffective treatments for metastatic cancer and metastases usuallyportend a poor outcome resulting in death. However, metastatic cancersare usually well vascularized and the delivery of anti-cancer antibodiesby red blood cells can have the effect of concentrating the antibodiesat the site of the tumor. Even prior to metastases, most cancer cellsare dependent on the host's blood supply for their survival and ananti-cancer antibody conjugated to red blood cells can be effectiveagainst in situ tumors as well. Alternatively, the antibodies may beconjugated to other hematogenous cells, e.g. lymphocytes, macrophages,monocytes, natural killer cells, etc.

There are five classes of antibodies and each is associated with afunction that is conferred by its heavy chain. It is generally thoughtthat cancer cell killing by naked antibodies are mediated either throughantibody dependent cellular cytotoxicity (ADCC) or complement dependentcytotoxicity (CDC). For example murine IgM and IgG2a antibodies canactivate human complement by binding the C-1 component of the complementsystem thereby activating the classical pathway of complement activationwhich can lead to tumor lysis. For human antibodies the most effectivecomplement activating antibodies are generally IgM and IgG1. Murineantibodies of the IgG2a and IgG3 isotype are effective at recruitingcytotoxic cells that have Fc receptors which will lead to cell killingby monocytes, macrophages, granulocytes and certain lymphocytes. Humanantibodies of both the IgG1 and IgG3 isotype mediate ADCC.

The cytotoxicity mediated through the Fc region requires the presence ofeffector cells, their corresponding receptors, or proteins e.g. NKcells, T cells and complement. In the absence of these effectormechanisms, the Fc portion of an antibody is inert. The Fc portion of anantibody may confer properties that affect the pharmacokinetics of anantibody in vivo, but in vitro this is not operative.

The cytotoxicity assays under which we test the antibodies do not haveany of the effector mechanisms present, and are carried out in vitro.These assays do not have effector cells (NK, Macrophages, or T-cells) orcomplement present. Since these assays are completely defined by what isadded together, each component can be characterized. The assays usedherein contain only target cells, media and sera. The target cells donot have effector functions since they are cancer cells or fibroblasts.Without exogenous cells which have effector function properties there isno cellular elements that have this function. The media does not containcomplement or any cells. The sera used to support the growth of thetarget cells do not have complement activity as disclosed by thevendors. Furthermore, in our own labs we have verified the absence ofcomplement activity in the sera used. Therefore, our work evidences thefact that the effects of the antibodies are due entirely to the effectsof the antigen binding which is mediated through the Fab. Effectively,the target cells are seeing and interacting with only the Fab, sincethey do not have receptors for the Fc. Although the hybridoma issecreting complete immunoglobulin which was tested with the targetcells, the only part of the immunoglobulin that interacts with the cellsare the Fab, which act as antigen binding fragments.

With respect to the instantly claimed antibodies and antigen bindingfragments, the application, as filed, has demonstrated cellularcytotoxicity as evidenced by the data in FIG. 1. As pointed out above,and as herein confirmed via objective evidence, this effect was entirelydue to binding by the Fab to the tumor cells.

Ample evidence exists in the art of antibodies mediating cytotoxicitydue to direct binding of the antibody to the target antigen independentof effector mechanisms recruited by the Fc. The best evidence for thisis in vitro experiments which do not have supplemental cells, orcomplement (to formally exclude those mechanisms). These types ofexperiments have been carried out with complete immunoglobulin, or withantigen binding fragments such as F(ab)′2 fragments. In these types ofexperiments, antibodies or antigen binding fragments can directly induceapoptosis of target cells such as in the case of anti-Her2 and anti-EGFRantibodies, both of which have been approved by the US FDA for marketingin cancer therapy.

Another possible mechanism of antibody mediated cancer killing may bethrough the use of antibodies that function to catalyze the hydrolysisof various chemical bonds in the cell membrane and its associatedglycoproteins or glycolipids, so-called catalytic antibodies.

There are three additional mechanisms of antibody-mediated cancer cellkilling. The first is the use of antibodies as a vaccine to induce thebody to produce an immune response against the putative antigen thatresides on the cancer cell. The second is the use of antibodies totarget growth receptors and interfere with their function or to downregulate that receptor so that its function is effectively lost. Thethird is the effect of such antibodies on direct ligation of cellsurface moieties that may lead to direct cell death, such as ligation ofdeath receptors such as TRAIL R1 or TRAIL R2, or integrin molecules suchas alpha V beta 3 and the like.

The clinical utility of a cancer drug is based on the benefit of thedrug under an acceptable risk profile to the patient. In cancer therapysurvival has generally been the most sought after benefit, however thereare a number of other well-recognized benefits in addition to prolonginglife. These other benefits, where treatment does not adversely affectsurvival, include symptom palliation, protection against adverse events,prolongation in time to recurrence or disease-free survival, andprolongation in time to progression. These criteria are generallyaccepted and regulatory bodies such as the U.S. Food and DrugAdministration (F.D.A.) approve drugs that produce these benefits(Hirschfeld et al. Critical Reviews in Oncology/Hematolgy 42:137-1432002). In addition to these criteria it is well recognized that thereare other endpoints that may presage these types of benefits. In part,the accelerated approval process granted by the U.S. F.D.A. acknowledgesthat there are surrogates that will likely predict patient benefit. Asof year-end 2003, there have been sixteen drugs approved under thisprocess, and of these, four have gone on to full approval, i.e.,follow-up studies have demonstrated direct patient benefit as predictedby surrogate endpoints. One important endpoint for determining drugeffects in solid tumors is the assessment of tumor burden by measuringresponse to treatment (Therasse et al. Journal of the National CancerInstitute 92(3):205-216 2000). The clinical criteria (RECIST criteria)for such evaluation have been promulgated by Response EvaluationCriteria in Solid Tumors Working Group, a group of international expertsin cancer. Drugs with a demonstrated effect on tumor burden, as shown byobjective responses according to RECIST criteria, in comparison to theappropriate control group tend to, ultimately, produce direct patientbenefit. In the pre-clinical setting tumor burden is generally morestraightforward to assess and document. In that pre-clinical studies canbe translated to the clinical setting, drugs that produce prolongedsurvival in pre-clinical models have the greatest anticipated clinicalutility. Analogous to producing positive responses to clinicaltreatment, drugs that reduce tumor burden in the pre-clinical settingmay also have significant direct impact on the disease. Althoughprolongation of survival is the most sought after clinical outcome fromcancer drug treatment, there are other benefits that have clinicalutility and it is clear that tumor burden reduction, which may correlateto a delay in disease progression, extended survival or both, can alsolead to direct benefits and have clinical impact (Eckhardt et al.Developmental Therapeutics: Successes and Failures of Clinical TrialDesigns of Targeted Compounds; ASCO Educational Book, 39^(th) AnnualMeeting, 2003, pages 209-219).

The present invention describes the development and use of AR52A301.5identified by, its effect, in a cytotoxic assay, in non-established andestablished tumor growth in animal models and in prolonging survivaltime in those suffering from cancerous disease. This inventionrepresents an advance in the field of cancer treatment in that itdescribes, for the first time, reagents that bind specifically to anepitope or epitopes present on the target molecule, TROP-2, and thatalso have in vitro cytotoxic properties, as a naked antibody, againstmalignant tumor cells but not normal cells, and which also directlymediate, as a naked antibody, inhibition of tumor growth and extensionof survival in in vivo models of human cancer. This is an advance inrelation to any other previously described anti-TROP-2 antibody, sincenone have been shown to have similar properties. It also provides anadvance in the field since it clearly demonstrates, and for the firsttime, the direct involvement of TROP-2 in events associated with growthand development of certain types of tumors. It also represents anadvance in cancer therapy since it has the potential to display similaranti-cancer properties in human patients. A further advance is thatinclusion of these antibodies in a library of anti-cancer antibodieswill enhance the possibility of targeting tumors expressing differentantigen markers by determination of the appropriate combination ofdifferent anti-cancer antibodies, to find the most effective intargeting and inhibiting growth and development of the tumors.

In all, this invention teaches the use of the AR52A301.5 antigen as atarget for a therapeutic agent, that when administered can reduce thetumor burden of a cancer expressing the antigen in a mammal, and canalso lead to a prolonged survival of the treated mammal. This inventionalso teaches the use of CDMAB (AR52A301.5), and its derivatives, andantigen binding fragments thereof, and cellular cytotoxicity inducingligands thereof to target their antigen to reduce the tumor burden of acancer expressing the antigen in a mammal, and lead to prolongedsurvival of the treated mammal. Furthermore, this invention also teachesthe use of detecting the AR52A301.5 antigen in cancerous cells that canbe useful for the diagnosis, prediction of therapy, and prognosis ofmammals bearing tumors that express this antigen.

Accordingly, it is an objective of the invention to utilize a method forproducing cancerous disease modifying antibodies (CDMAB) raised againstcancerous cells derived from a particular individual, or one or moreparticular cancer cell lines, which CDMAB are cytotoxic with respect tocancer cells while simultaneously being relatively non-toxic tonon-cancerous cells, in order to isolate hybridoma cell lines and thecorresponding isolated monoclonal antibodies and antigen bindingfragments thereof for which said hybridoma cell lines are encoded.

It is an additional objective of the invention to teach cancerousdisease modifying antibodies, ligands and antigen binding fragmentsthereof.

It is a further objective of the instant invention to produce cancerousdisease modifying antibodies whose cytotoxicity is mediated throughantibody dependent cellular toxicity.

It is yet an additional objective of the instant invention to producecancerous disease modifying antibodies whose cytotoxicity is mediatedthrough complement dependent cellular toxicity.

It is still a further objective of the instant invention to producecancerous disease modifying antibodies whose cytotoxicity is a functionof their ability to catalyze hydrolysis of cellular chemical bonds.

A still further objective of the instant invention is to producecancerous disease modifying antibodies which are useful for in a bindingassay for diagnosis, prognosis, and monitoring of cancer.

Other objects and advantages of this invention will become apparent fromthe following description wherein are set forth, by way of illustrationand example, certain embodiments of this invention.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 compares the percentage cytotoxicity and binding levels of thehybridoma supernatants against cell lines OCC-1, OVCAR-3 and CCD-27sk.

FIG. 2 tabulates binding of AR52A301.5 and the anti-EGFR antibodycontrol to cancer and normal cell lines. The data is presented as themean fluorescence intensity as a fold increase above isotype control.

FIG. 3 includes representative FACS histograms of AR52A301.5 andanti-EGFR antibodies directed against several cancer and non-cancer celllines.

FIG. 4 demonstrates the effect of AR52A301.5 on tumor growth in aprophylactic BxPC-3 pancreatic cancer model. The vertical lines indicatethe period during which the antibody was administered. Data pointsrepresent the mean+/−SEM.

FIG. 5 demonstrates the effect of AR52A301.5 on body weight in aprophylactic BxPC-3 pancreatic cancer model. Data points represent themean+/−SEM.

FIG. 6 demonstrates the effect of AR52A301.5 on tumor growth in anestablished BxPC-3 pancreatic cancer model. The vertical lines indicatethe period during which the antibody was administered. Data pointsrepresent the mean+/−SEM.

FIG. 7 demonstrates the effect of AR52A301.5 on body weight in anestablished BxPC-3 pancreatic cancer model. Data points represent themean+/−SEM.

FIG. 8 demonstrates the effect of AR52A301.5 on tumor growth in aprophylactic PL45 pancreatic cancer model. The vertical lines indicatethe period during which the antibody was administered. Data pointsrepresent the mean+/−SEM.

FIG. 9 demonstrates the effect of AR52A301.5 on survival in aprophylactic PL45 pancreatic cancer model.

FIG. 10 demonstrates the effect of AR52A301.5 on body weight in aprophylactic PL45 pancreatic cancer model. Data points represent themean+/−SEM.

FIG. 11 demonstrates the effect of AR52A301.5 on tumor growth in aprophylactic PC-3 prostate cancer model. The vertical lines indicate theperiod during which the antibody was administered. Data points representthe mean+/−SEM.

FIG. 12 demonstrates the effect of AR52A301.5 on survival in aprophylactic PC-3 prostate cancer model.

FIG. 13 demonstrates the effect of AR52A301.5 on body weight in aprophylactic PC-3 prostate cancer model. Data points represent themean+/−SEM.

FIG. 14 demonstrates the effect of AR52A301.5 on tumor growth in aprophylactic Colo 205 colon cancer model. The vertical lines indicatethe period during which the antibody was administered. Data pointsrepresent the mean+/−SEM.

FIG. 15 demonstrates the effect of AR52A301.5 on body weight in aprophylactic Colo 205 colon cancer model. Data points represent themean+/−SEM.

FIG. 16. Western blot of samples from the total membrane fraction ofMDA-MB-231 cells (lane 1) and of whole cell lysates from PC-3 (lane 2)and CCD-27sk (lane 3) cell lines. Blots were probed with the AR47A6.4.2and AR52A301.5. Molecular weight markers are indicated on the left.

FIG. 17. Western blot of an immunocomplex prepared byimmunoprecipitation with AR47A6.4.2 (lane 1) and with an isotype control(lane 2), from the total membrane fraction of the MDA-MB-231 cell line.Antibody-conjugated Protein G-Sepharose beads not incubated with totalmembrane fraction of MDA-MB-231 were also used as negative controls(lanes 3 and 4). Replicate blots were probed either with the antibodiesAR47A6.4.2, AR52A301.5 (hybridoma supernatant), IgG2a isotype control orwithout primary antibody. Samples were analyzed by SDS-PAGEelectrophoresis under reducing and non-reducing conditions. Molecularweight markers are indicated on the left.

FIG. 18. Western blot of recombinant human TROP-2. Individual lanes wereprobed with the AR52A301.5 (lane 1) and AR47A6.4.2 (lane 3), and withisotype control antibodies (lanes 2 and 4).

FIG. 19. MDA-MB-231 membrane proteins (MB-231) and recombinant humanTROP-2 (rhTROP-2) glycosylated (G) and deglycosylated (D) underdenaturing and non-denaturing conditions, probed with AR52A301.5.

FIG. 20. MDA-MB-231 membrane proteins (MB-231) and recombinant humanTROP-2 (rhTROP-2) glycosylated (G) and deglycosylated (D) underdenaturing and non-denaturing conditions, probed with anti-human TROP-2.

FIG. 21. MDA-MB-231 membrane proteins (MB-231) and recombinant humanTROP-2 (rhTROP-2) glycosylated (G) and deglycosylated (D) underdenaturing and non-denaturing conditions, probed with 1B7.11 IgG1isotype control.

FIG. 22. Western blot of recombinant human TROP-2 probed with differentprimary antibody solutions. Lanes 3 to 7 were probed with biotinylatedAR52A301.5 mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL,500 microgram/mL and 1000 microgram/mL of non-biotinylated AR52A301.5respectively. Lanes 9 to 13 were probed with biotinylated AR52A301.5mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500microgram/mL and 1000 microgram/mL of non-biotinylated AR47A6.4.2respectively. Lanes 15 to 19 were probed with biotinylated AR52A301.5mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500microgram/mL and 1000 microgram/mL of non-biotinylated 8A3B.6respectively. Lanes 8 and 14 were incubated with negative controlsolution and lane 8 was not incubated in secondary solution. Lanes 1, 2and 20 were incubated with TBST only.

FIG. 23. Western blot of recombinant human TROP-2 probed with differentprimary antibody solutions. Lanes 3 to 7 were probed with biotinylatedAR47A6.4.2 mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL,500 microgram/mL and 1000 microgram/mL of non-biotinylated AR52A301.5respectively. Lanes 9 to 13 were probed with biotinylated AR47A6.4.2mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500microgram/mL and 1000 microgram/mL of non-biotinylated AR47A6.4.2respectively. Lanes 15 to 19 were probed with biotinylated AR47A6.4.2mixed with 0.5 microgram/mL, 5 microgram/mL, 50 microgram/mL, 500microgram/mL and 1000 microgram/mL of non-biotinylated 1B7.11respectively. Lanes 8 and 14 were incubated with negative controlsolution and lane 8 was not incubated in secondary solution. Lanes 1, 2and 20 were incubated with TBST only.

FIG. 24 tabulates an IHC comparison of AR52A301.5 versus positive andnegative controls on a normal human tissue micro array.

FIG. 25. Representative micrographs showing the binding pattern onspleen tissue obtained with AR52A301.5 (A) or the isotype controlantibody (B) and on brain tissue obtained with AR52A301.5 (C) or theisotype control antibody (D) from a normal human tissue microarray.Magnification is 200×.

FIG. 26 tabulates an IHC comparison of AR52A301.5 on various human tumorand normal tissue sections from different normal human tissue microarrays.

FIG. 27. Representative micrographs showing the binding pattern onbreast tumor tissue obtained with AR52A301.5 (A) or the isotype controlantibody (B) and on prostate tumor tissue obtained with AR52A301.5 (C)or the isotype control antibody (D) and on pancreatic tumor tissueobtained with AR52A301.5 (E) or the isotype control antibody (F) fromvarious human tumor tissue microarrays. Magnification is 200×.

FIG. 28 tabulates an IHC comparison of AR52A301.5 on various human andother species normal tissue sections from different normal speciestissue micro arrays.

FIG. 29. Representative micrographs showing the binding pattern onnormal human kidney tissue obtained with AR52A301.5 (A) or the isotypecontrol antibody (B) and on normal cynomolgus kidney tissue obtainedwith AR52A301.5 (C) or the isotype control antibody (D) and on normalrhesus tissue obtained with AR52A301.5 (E) or the isotype controlantibody (F) from various multi-species tissue microarrays.Magnification is 200×.

FIG. 30. Sequences of all oligonucleotide primers (SEQ ID NOS:9-46) usedin the murine sequence determination of AR52A301.5.

FIG. 31. Agarose gel of the RT/PCR amplification of AR52A301.5 V_(H) andV_(L) regions.

FIG. 32. Agarose gel of the PCR colony screen of AR52A301.5 V_(H)-B,V_(H)-E and V_(L)-G.

FIG. 33. AR52A301.5 V_(L) amino acid sequence (SEQ ID NO:8).

FIG. 34. AR52A301.5 V_(H) amino acid sequence (SEQ ID NO:7).

DETAILED DESCRIPTION OF THE INVENTION

In general, the following words or phrases have the indicated definitionwhen used in the summary, description, examples, and claims.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies, de-immunized, murine, chimericor humanized antibodies), antibody compositions with polyepitopicspecificity, single-chain antibodies, diabodies, triabodies,immunoconjugates and antibody fragments (see below).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma (murine orhuman) method first described by Kohler et al., Nature, 256:495 (1975),or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). The “monoclonal antibodies” may also be isolated from phageantibody libraries using the techniques described in Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991), for example.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include less than full length antibodies,Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; single-chain antibodies, single domainantibody molecules, fusion proteins, recombinant proteins andmultispecific antibodies formed from antibody fragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof. Preferably, the intactantibody has one or more effector functions.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five-major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., Eur. J. Immunol. 24:2429 (1994)).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues2632 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, whose name reflects its ability to crystallize readily.Pepsin treatment yields an F(ab′)₂ fragment that has two antigen-bindingsites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site. The Fab fragment alsocontains the constant domain of the light chain and the first constantdomain (CH I) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear at least onefree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

The term “triabodies” or “trivalent trimers” refers to the combinationof three single chain antibodies. Triabodies are constructed with theamino acid terminus of a V_(L) or V_(H) domain, i.e., without any linkersequence. A triabody has three Fv heads with the polypeptides arrangedin a cyclic, head-to-tail fashion. A possible conformation of thetriabody is planar with the three binding sites located in a plane at anangle of 120 degrees from one another. Triabodies can be monospecific,bispecific or trispecific.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

An antibody “which binds” an antigen of interest, e.g. TROP-2 antigen,is one capable of binding that antigen with sufficient affinity suchthat the antibody is useful as a therapeutic or diagnostic agent intargeting a cell expressing the antigen. Where the antibody is one whichbinds TROP-2, it will usually preferentially bind TROP-2 as opposed toother receptors, and does not include incidental binding such asnon-specific Fc contact, or binding to post-translational modificationscommon to other antigens and may be one which does not significantlycross-react with other proteins. Methods, for the detection of anantibody that binds an antigen of interest, are well known in the artand can include but are not limited to assays such as FACS, cell ELISAand Western blot.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably, and all such designations include progeny. Itis also understood that all progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included. It will be clear from thecontext where distinct designations are intended.

“Treatment or treating” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathologic condition or disorder.Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented. Hence, the mammal to be treated herein may have beendiagnosed as having the disorder or may be predisposed or susceptible tothe disorder.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth or death. Examples of cancer include, but arenot limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia orlymphoid malignancies. More particular examples of such cancers includesquamous cell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, as well as head and neckcancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, carnomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Aventis, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, mice, SCID or nude mice or strains of mice,domestic and farm animals, and zoo, sports, or pet animals, such assheep, dogs, horses, cats, cows, etc. Preferably, the mammal herein ishuman.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP 266,032, published 4 May1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14:5399-5407, 1986. They are thenpurified on polyacrylamide gels.

In accordance with the present invention, “humanized” and/or “chimeric”forms of non-human (e.g. murine) immunoglobulins refer to antibodieswhich contain specific chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which results in thedecrease of a human anti-mouse antibody (HAMA), human anti-chimericantibody (HACA) or a human anti-human antibody (HAHA) response, comparedto the original antibody, and contain the requisite portions (e.g.CDR(s), antigen binding region(s), variable domain(s) and so on) derivedfrom said non-human immunoglobulin, necessary to reproduce the desiredeffect, while simultaneously retaining binding characteristics which arecomparable to said non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from the complementarity determining regions (CDRs) ofthe recipient antibody are replaced by residues from the CDRs of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human FR residues. Furthermore, the humanizedantibody may comprise residues which are found neither in the recipientantibody nor in the imported CDR or FR sequences. These modificationsare made to further refine and optimize antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR residues are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

“De-immunized” antibodies are immunoglobulins that are non-immunogenic,or less immunogenic, to a given species. De-immunization can be achievedthrough structural alterations to the antibody. Any de-immunizationtechnique known to those skilled in the art can be employed. Onesuitable technique for de-immunizing antibodies is described, forexample, in WO 00/34317 published Jun. 15, 2000.

An antibody which induces “apoptosis” is one which induces programmedcell death by any means, illustrated by but not limited to binding ofannexin V, caspase activity, fragmentation of DNA, cell shrinkage,dilation of endoplasmic reticulum, cell fragmentation, and/or formationof membrane vesicles (called apoptotic bodies).

As used herein “antibody induced cytotoxicity” is understood to mean thecytotoxic effect derived from the hybridoma supernatant or antibodyproduced by the hybridoma deposited with the IDAC as accession number141205-03 which effect is not necessarily related to the degree ofbinding.

Throughout the instant specification, hybridoma cell lines, as well asthe isolated monoclonal antibodies which are produced therefrom, arealternatively referred to by their internal designation, AR52A301.5 orDepository Designation, IDAC 141205-03.

As used herein “antibody-ligand” includes a moiety which exhibitsbinding specificity for at least one epitope of the target antigen, andwhich may be an intact antibody molecule, antibody fragments, and anymolecule having at least an antigen-binding region or portion thereof(i.e., the variable portion of an antibody molecule), e.g., an Fvmolecule, Fab molecule, Fab′ molecule, F(ab′).sub.2 molecule, abispecific antibody, a fusion protein, or any genetically engineeredmolecule which specifically recognizes and binds at least one epitope ofthe antigen bound by the isolated monoclonal antibody produced by thehybridoma cell line designated as IDAC 141205-03 (the IDAC 141205-03antigen).

As used herein “cancerous disease modifying antibodies” (CDMAB) refersto monoclonal antibodies which modify the cancerous disease process in amanner which is beneficial to the patient, for example by reducing tumorburden or prolonging survival of tumor bearing individuals, andantibody-ligands thereof.

A “CDMAB related binding agent”, in its broadest sense, is understood toinclude, but is not limited to, any form of human or non-humanantibodies, antibody fragments, antibody ligands, or the like, whichcompetitively bind to at least one CDMAB target epitope.

A “competitive binder” is understood to include any form of human ornon-human antibodies, antibody fragments, antibody ligands, or the likewhich has binding affinity for at least one CDMAB target epitope.

Tumors to be treated include primary tumors and metastatic tumors, aswell as refractory tumors. Refractory tumors include tumors that fail torespond or are resistant to treatment with chemotherapeutic agentsalone, antibodies alone, radiation alone or combinations thereof.Refractory tumors also encompass tumors that appear to be inhibited bytreatment with such agents but recur up to five years, sometimes up toten years or longer after treatment is discontinued.

Tumors that can be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors.Examples of solid tumors, which can be accordingly treated, includebreast carcinoma, lung carcinoma, colorectal carcinoma, pancreaticcarcinoma, glioma and lymphoma. Some examples of such tumors includeepidermoid tumors, squamous tumors, such as head and neck tumors,colorectal tumors, prostate tumors, breast tumors, lung tumors,including small cell and non-small cell lung tumors, pancreatic tumors,thyroid tumors, ovarian tumors, and liver tumors. Other examples includeKaposi's sarcoma, CNS neoplasms, neuroblastomas; capillaryhemangioblastomas, meningiomas and cerebral metastases, melanoma,gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma,glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma.

As used herein “antigen-binding region” means a portion of the moleculewhich recognizes the target antigen.

As used herein “competitively inhibits” means being able to recognizeand bind a determinant site to which the monoclonal antibody produced bythe hybridoma cell line designated as IDAC 141205-03, (the IDAC141205-03 antibody) is directed using conventional reciprocal antibodycompetition assays. (Belanger L., Sylvestre C. and Dufour D. (1973),Enzyme linked immunoassay for alpha fetoprotein by competitive andsandwich procedures. Clinica Chimica Acta 48, 15).

As used herein “target antigen” is the IDAC 141205-03 antigen orportions thereof.

As used herein, an “immunoconjugate” means any molecule or CDMAB such asan antibody chemically or biologically linked to cytotoxins, radioactiveagents, cytokines, interferons, target or reporter moieties, enzymes,toxins, anti-tumor drugs or therapeutic agents. The antibody or CDMABmay be linked to the cytotoxin, radioactive agent, cytokine, interferon,target or reporter moiety, enzyme, toxin, anti-tumor drug or therapeuticagent at any location along the molecule so long as it is able to bindits target. Examples of immunoconjugates include antibody toxin chemicalconjugates and antibody-toxin fusion proteins.

Radioactive agents suitable for use as anti-tumor agents are known tothose skilled in the art. For example, 131I or 211At is used. Theseisotopes are attached to the antibody using conventional techniques(e.g. Pedley et al., Br. J. Cancer 68, 69-73 (1993)). Alternatively, theanti-tumor agent which is attached to the antibody is an enzyme whichactivates a prodrug. A prodrug may be administered which will remain inits inactive form until it reaches the tumor site where it is convertedto its cytotoxin form once the antibody complex is administered. Inpractice, the antibody-enzyme conjugate is administered to the patientand allowed to localize in the region of the tissue to be treated. Theprodrug is then administered to the patient so that conversion to thecytotoxic drug occurs in the region of the tissue to be treated.Alternatively, the anti-tumor agent conjugated to the antibody is acytokine such as interleukin-2 (IL-2), interleukin-4 (IL-4) or tumornecrosis factor alpha (TNF-α). The antibody targets the cytokine to thetumor so that the cytokine mediates damage to or destruction of thetumor without affecting other tissues. The cytokine is fused to theantibody at the DNA level using conventional recombinant DNA techniques.Interferons may also be used.

As used herein, a “fusion protein” means any chimeric protein wherein anantigen binding region is connected to a biologically active molecule,e.g., toxin, enzyme, fluorescent proteins, luminescent marker,polypeptide tag, cytokine, interferon, target or reporter moiety orprotein drug.

The invention further contemplates CDMAB of the present invention towhich target or reporter moieties are linked. Target moieties are firstmembers of binding pairs. Anti-tumor agents, for example, are conjugatedto second members of such pairs and are thereby directed to the sitewhere the antigen-binding protein is bound. A common example of such abinding pair is avidin and biotin. In a preferred embodiment, biotin isconjugated to the target antigen of the CDMAB of the present invention,and thereby provides a target for an anti-tumor agent or other moietywhich is conjugated to avidin or streptavidin. Alternatively, biotin oranother such moiety is linked to the target antigen of the CDMAB of thepresent invention and used as a reporter, for example in a diagnosticsystem where a detectable signal-producing agent is conjugated to avidinor streptavidin.

Detectable signal-producing agents are useful in vivo and in vitro fordiagnostic purposes. The signal producing agent produces a measurablesignal which is detectable by external means, usually the measurement ofelectromagnetic radiation. For the most part, the signal producing agentis an enzyme or chromophore, or emits light by fluorescence,phosphorescence or chemiluminescence. Chromophores include dyes whichabsorb light in the ultraviolet or visible region, and can be substratesor degradation products of enzyme catalyzed reactions.

Moreover, included within the scope of the present invention is use ofthe present CDMAB in vivo and in vitro for investigative or diagnosticmethods, which are well known in the art. In order to carry out thediagnostic methods as contemplated herein, the instant invention mayfurther include kits, which contain CDMAB of the present invention. Suchkits will be useful for identification of individuals at risk forcertain type of cancers by detecting over-expression of the CDMAB'starget antigen on cells of such individuals.

Diagnostic Assay Kits

It is contemplated to utilize the CDMAB of the present invention in theform of a diagnostic assay kit for determining the presence of a tumor.The tumor will generally be detected in a patient based on the presenceof one or more tumor-specific antigens, e.g. proteins and/orpolynucleotides which encode such proteins in a biological sample, suchas blood, sera, urine and/or tumor biopsies, which samples will havebeen obtained from the patient.

The proteins function as markers which indicate the presence or absenceof a particular tumor, for example a colon, breast, lung or prostatetumor. It is further contemplated that the antigen will have utility forthe detection of other cancerous tumors. Inclusion in the diagnosticassay kits of binding agents comprised of CDMABs of the presentinvention, or CDMAB related binding agents, enables detection of thelevel of antigen that binds to the agent in the biological sample.Polynucleotide primers and probes may be used to detect the level ofmRNA encoding a tumor protein, which is also indicative of the presenceor absence of a cancer. In order for the binding assay to be diagnostic,data will have been generated which correlates statistically significantlevels of antigen, in relation to that present in normal tissue, so asto render the recognition of binding definitively diagnostic for thepresence of a cancerous tumor. It is contemplated that a plurality offormats will be useful for the diagnostic assay of the presentinvention, as are known to those of ordinary skill in the art, for usinga binding agent to detect polypeptide markers in a sample. For example,as illustrated in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988. Further contemplated are any and allcombinations, permutations or modifications of the afore-describeddiagnostic assay formats.

The presence or absence of a cancer in a patient will typically bedetermined by (a) contacting a biological sample obtained from a patientwith a binding agent; (b) detecting in the sample a level of polypeptidethat binds to the binding agent; and (c) comparing the level ofpolypeptide with a predetermined cut-off value.

In an illustrative embodiment, it is contemplated that the assay willinvolve the use of a CDMAB based binding agent immobilized on a solidsupport to bind to and remove the polypeptide from the remainder of thesample. The bound polypeptide may then be detected using a detectionreagent that contains a reporter group and specifically binds to thebinding agent/polypeptide complex. Illustrative detection reagents mayinclude a CDMAB based binding agent that specifically binds to thepolypeptide or an antibody or other agent that specifically binds to thebinding agent, such as an anti-immunoglobulin, protein G, protein A or alectin. In an alternative embodiment, it is contemplated that acompetitive assay may be utilized, in which a polypeptide is labeledwith a reporter group and allowed to bind to the immobilized bindingagent after incubation of the binding agent with the sample. Indicativeof the reactivity of the sample with the immobilized binding agent, isthe extent to which components of the sample inhibit the binding of thelabeled polypeptide to the binding agent. Suitable polypeptides for usewithin such assays include full length tumor-specific proteins and/orportions thereof, to which the binding agent has binding affinity.

The diagnostic kit will be provided with a solid support which may be inthe form of any material known to those of ordinary skill in the art towhich the protein may be attached. Suitable examples may include a testwell in a microtiter plate or a nitrocellulose or other suitablemembrane. Alternatively, the support may be a bead or disc, such asglass, fiberglass, latex or a plastic material such as polystyrene orpolyvinylchloride. The support may also be a magnetic particle or afiber optic sensor, such as those disclosed, for example, in U.S. Pat.No. 5,359,681.

It is contemplated that the binding agent will be immobilized on thesolid support using a variety of techniques known to those of skill inthe art, which are amply described in the patent and scientificliterature. The term “immobilization” refers to both noncovalentassociation, such as adsorption, and covalent attachment, which, in thecontext of the present invention, may be a direct linkage between theagent and functional groups on the support, or may be a linkage by wayof a cross-linking agent. In a preferred, albeit non-limitingembodiment, immobilization by adsorption to a well in a microtiter plateor to a membrane is preferable. Adsorption may be achieved by contactingthe binding agent, in a suitable buffer, with the solid support for asuitable amount of time. The contact time may vary with temperature, andwill generally be within a range of between about 1 hour and about 1day.

Covalent attachment of binding agent to a solid support would ordinarilybe accomplished by first reacting the support with a bifunctionalreagent that will react with both the support and a functional group,such as a hydroxyl or amino group, on the binding agent. For example,the binding agent may be covalently attached to supports having anappropriate polymer coating using benzoquinone or by condensation of analdehyde group on the support with an amine and an active hydrogen onthe binding partner (see, e.g., Pierce Immunotechnology Catalog andHandbook, 1991, at A12 A13).

It is further contemplated that the diagnostic assay kit will take theform of a two-antibody sandwich assay. This assay may be performed byfirst contacting an antibody, e.g. the instantly disclosed CDMAB thathas been immobilized on a solid support, commonly the well of amicrotiter plate, with the sample, such that polypeptides within thesample are allowed to bind to the immobilized antibody. Unbound sampleis then removed from the immobilized polypeptide-antibody complexes anda detection reagent (preferably a second antibody capable of binding toa different site on the polypeptide) containing a reporter group isadded. The amount of detection reagent that remains bound to the solidsupport is then determined using a method appropriate for the specificreporter group.

In a specific embodiment, it is contemplated that once the antibody isimmobilized on the support as described above, the remaining proteinbinding sites on the support will be blocked, via the use of anysuitable blocking agent known to those of ordinary skill in the art,such as bovine serum albumin or Tween 20™ (Sigma Chemical Co., St.Louis, Mo.). The immobilized antibody would then be incubated with thesample, and polypeptide would be allowed to bind to the antibody. Thesample could be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) would be selected tocorrespond to a period of time sufficient to detect the presence ofpolypeptide within a sample obtained from an individual with thespecifically selected tumor. Preferably, the contact time is sufficientto achieve a level of binding that is at least about 95 percent of thatachieved at equilibrium between bound and unbound polypeptide. Those ofordinary skill in the art will recognize that the time necessary toachieve equilibrium may be readily determined by assaying the level ofbinding that occurs over a period of time.

It is further contemplated that unbound sample would then be removed bywashing the solid support with an appropriate buffer. The secondantibody, which contains a reporter group, would then be added to thesolid support. Incubation of the detection reagent with the immobilizedantibody-polypeptide complex would then be carried out for an amount oftime sufficient to detect the bound polypeptide. Subsequently, unbounddetection reagent would then be removed and bound detection reagentwould be detected using the reporter group. The method employed fordetecting the reporter group is necessarily specific to the type ofreporter group selected, for example for radioactive groups,scintillation counting or autoradiographic methods are generallyappropriate. Spectroscopic methods may be used to detect dyes,luminescent groups and fluorescent groups. Biotin may be detected usingavidin, coupled to a different reporter group (commonly a radioactive orfluorescent group or an enzyme). Enzyme reporter groups may generally bedetected by the addition of substrate (generally for a specific periodof time), followed by spectroscopic or other analysis of the reactionproducts.

In order to utilize the diagnostic assay kit of the present invention todetermine the presence or absence of a cancer, such as prostate cancer,the signal detected from the reporter group that remains bound to thesolid support would generally be compared to a signal that correspondsto a predetermined cut-off value. For example, an illustrative cut-offvalue for the detection of a cancer may be the average mean signalobtained when the immobilized antibody is incubated with samples frompatients without the cancer. In general, a sample generating a signalthat is about three standard deviations above the predetermined cut-offvalue would be considered positive for the cancer. In an alternateembodiment, the cut-off value might be determined by using a ReceiverOperator Curve, according to the method of Sackett et al., ClinicalEpidemiology. A Basic Science for Clinical Medicine, Little Brown andCo., 1985, p. 106-7. In such an embodiment, the cut-off value could bedetermined from a plot of pairs of true positive rates (i.e.,sensitivity) and false positive rates (100 percent-specificity) thatcorrespond to each possible cut-off value for the diagnostic testresult. The cut-off value on the plot that is the closest to the upperleft-hand corner (i.e., the value that encloses the largest area) is themost accurate cut-off value, and a sample generating a signal that ishigher than the cut-off value determined by this method may beconsidered positive. Alternatively, the cut-off value may be shifted tothe left along the plot, to minimize the false positive rate, or to theright, to minimize the false negative rate. In general, a samplegenerating a signal that is higher than the cut-off value determined bythis method is considered positive for a cancer.

It is contemplated that the diagnostic assay enabled by the kit will beperformed in either a flow-through or strip test format, wherein thebinding agent is immobilized on a membrane, such as nitrocellulose. Inthe flow-through test, polypeptides within the sample bind to theimmobilized binding agent as the sample passes through the membrane. Asecond, labeled binding agent then binds to the bindingagent-polypeptide complex as a solution containing the second bindingagent flows through the membrane. The detection of bound second bindingagent may then be performed as described above. In the strip testformat, one end of the membrane to which binding agent is bound will beimmersed in a solution containing the sample. The sample migrates alongthe membrane through a region containing second binding agent and to thearea of immobilized binding agent. Concentration of the second bindingagent at the area of immobilized antibody indicates the presence of acancer. Generation of a pattern, such as a line, at the binding site,which can be read visually, will be indicative of a positive test. Theabsence of such a pattern indicates a negative result. In general, theamount of binding agent immobilized on the membrane is selected togenerate a visually discernible pattern when the biological samplecontains a level of polypeptide that would be sufficient to generate apositive signal in the two-antibody sandwich assay, in the formatdiscussed above. Preferred binding agents for use in the instantdiagnostic assay are the instantly disclosed antibodies, antigen-bindingfragments thereof, and any CDMAB related binding agents as hereindescribed. The amount of antibody immobilized on the membrane will beany amount effective to produce a diagnostic assay, and may range fromabout 25 nanograms to about 1 microgram. Typically such tests may beperformed with a very small amount of biological sample.

Additionally, the CDMAB of the present invention may be used in thelaboratory for research due to its ability to identify its targetantigen.

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

The present invention provides CDMAB (i.e., IDAC 141205-03 CDMAB) whichspecifically recognize and bind the IDAC 141205-03 antigen.

The CDMAB of the isolated monoclonal antibody produced by the hybridomadeposited with the IDAC as accession number 141205-03 may be in any formas long as it has an antigen-binding region which competitively inhibitsthe immunospecific binding of the isolated monoclonal antibody producedby hybridoma IDAC 141205-03 to its target antigen. Thus, any recombinantproteins (e.g., fusion proteins wherein the antibody is combined with asecond protein such as a lymphokine or a tumor inhibitory growth factor)having the same binding specificity as the IDAC 141205-03 antibody fallwithin the scope of this invention.

In one embodiment of the invention, the CDMAB is the IDAC 141205-03antibody.

In other embodiments, the CDMAB is an antigen binding fragment which maybe a Fv molecule (such as a single-chain Fv molecule), a Fab molecule, aFab′ molecule, a F(ab′)2 molecule, a fusion protein, a bispecificantibody, a heteroantibody or any recombinant molecule having theantigen-binding region of the IDAC 141205-03 antibody. The CDMAB of theinvention is directed to the epitope to which the IDAC 141205-03monoclonal antibody is directed.

The CDMAB of the invention may be modified, i.e., by amino acidmodifications within the molecule, so as to produce derivativemolecules. Chemical modification may also be possible. Modification bydirect mutation, methods of affinity maturation, phage display or chainshuffling may also be possible.

Affinity and specificity can be modified or improved by mutating CDRand/or phenylalanine tryptophan (FW) residues and screening for antigenbinding sites having the desired characteristics (e.g., Yang et al., J.Mol. Biol., (1995) 254: 392-403). One way is to randomize individualresidues or combinations of residues so that in a population ofotherwise identical antigen binding sites, subsets of from two to twentyamino acids are found at particular positions. Alternatively, mutationscan be induced over a range of residues by error prone PCR methods(e.g., Hawkins et al., J. Mol. Biol., (1992) 226: 889-96). In anotherexample, phage display vectors containing heavy and light chain variableregion genes can be propagated in mutator strains of E. coli (e.g., Lowet al., J. Mol. Biol., (1996) 250: 359-68). These methods of mutagenesisare illustrative of the many methods known to one of skill in the art.

Another manner for increasing affinity of the antibodies of the presentinvention is to carry out chain shuffling, where the heavy or lightchain are randomly paired with other heavy or light chains to prepare anantibody with higher affinity. The various CDRs of the antibodies mayalso be shuffled with the corresponding CDRs in other antibodies.

Derivative molecules would retain the functional property of thepolypeptide, namely, the molecule having such substitutions will stillpermit the binding of the polypeptide to the IDAC 141205-03 antigen orportions thereof.

These amino acid substitutions include, but are not necessarily limitedto, amino acid substitutions known in the art as “conservative”.

For example, it is a well-established principle of protein chemistrythat certain amino acid substitutions, entitled “conservative amino acidsubstitutions,” can frequently be made in a protein without alteringeither the conformation or the function of the protein.

Such changes include substituting any of isoleucine (I), valine (V), andleucine (L) for any other of these hydrophobic amino acids; asparticacid (D) for glutamic acid (E) and vice versa; glutamine (Q) forasparagine (N) and vice versa; and serine (S) for threonine (T) and viceversa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanineand valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

EXAMPLE 1 Hybridoma Production Hybridoma Cell Line AR52A301.5

The hybridoma cell line AR52A301.5 was deposited, in accordance with theBudapest Treaty, with the International Depository Authority of Canada(IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street,Winnipeg, Manitoba, Canada, R3E,3R2, on Dec. 14, 2005, under AccessionNumber 141205-03. In accordance with 37 CFR 1.808, the depositors assurethat all restrictions imposed on the availability to the public of thedeposited materials will be irrevocably removed upon the granting of apatent. The deposit will be replaced if the depository cannot dispenseviable samples.

To produce the hybridoma that produces the anti-cancer antibodiesAR52A301.5, a single cell suspension of frozen human ovarian tumortissue (endometroid adenocarcinoma; Genomics Collaborative, Cambridge,Mass.) was prepared in PBS. IMMUNEASY™ (Qiagen, Venlo, Netherlands)adjuvant was prepared for use by gentle mixing. Five to seven week oldBALB/c mice were immunized by injecting intraperitoneally, 2 millioncells in 50 microliters of the antigen-adjuvant. Recently preparedantigen-adjuvant was used to boost both the AR52A301.5 immunized miceintraperitoneally, 2 and 5 weeks after the initial immunization, withapproximately 2 million cells in 50-60 microliters. A spleen was usedfor fusion three days after the last immunization. The hybridomas wereprepared by fusing the isolated splenocytes with NSO-1 myeloma partners.The supernatants from the fusions were tested from subclones of thehybridomas.

To determine whether the antibodies secreted by the hybridoma cells areof the IgG or IgM isotype, an ELISA assay was employed. 100microliters/well of goat anti-mouse IgG+IgM (H+L) at a concentration of2.4 micrograms/mL in coating buffer (0.1 M carbonate/bicarbonate buffer,pH 9.2-9.6) at 4° C. was added to the ELISA plates overnight. The plateswere washed thrice in washing buffer (PBS+0.05 percent Tween-20). 100microliters/well blocking buffer (5 percent milk in wash buffer) wasadded to the plate for 1 hour at room temperature and then washed thricein washing buffer. 100 microliters/well of hybridoma supernatant wasadded and the plate incubated for 1 hour at room temperature. The plateswere washed thrice with washing buffer and 1/100,000 dilution of eithergoat anti-mouse IgG or IgM horseradish peroxidase conjugate (diluted inPBS containing 1 percent milk), 100 microliters/well, was added. Afterincubating the plate for 1 hour at room temperature the plate was washedthrice with washing buffer. 100 microliters/well of TMB solution wasincubated for 1-3 minutes at room temperature. The color reaction wasterminated by adding 50 microliters/well 2M H₂S0₄ and the plate was readat 450 nm with a Perkin-Elmer HTS7000 plate reader. As indicated in FIG.1, the AR52A301.5 hybridoma secreted primarily antibodies of the IgGisotype.

To determine the subclass of antibody secreted by the hybridoma cells,an isotyping experiment was performed using a Mouse Monoclonal AntibodyIsotyping Kit (HyCult Biotechnology, Frontstraat, Netherlands). 500microliters of buffer solution was added to the test strip containingrat anti-mouse subclass specific antibodies. 500 microliters ofhybridoma supernatant was added to the test tube, and submerged bygentle agitation. Captured mouse immunoglobulins were detected directlyby a second rat monoclonal antibody which is coupled to colloidparticles. The combination of these two proteins creates a visual signalused to analyse the isotype. The anti-cancer antibody AR52A301.5 is ofthe IgG1, kappa isotype.

After one round of limiting dilution, hybridoma supernatants were testedfor antibodies that bound to target cells in a cell ELISA assay. Twohuman ovarian cancer cell lines and 1 human normal skin cell line weretested: OCC-1, OVCAR-3 and CCD-27sk respectively. All cell lines, exceptfor OCC-1, were obtained from the American Type Tissue Collection (ATCC,Manassas, Va.). The OCC-1 ovarian cancer cell line was obtained from theOttawa Regional Cancer Center (Ottawa, ON).

The plated cells were fixed prior to use. The plates were washed thricewith PBS containing MgCl₂ and CaCl₂ at room temperature. 100 microlitersof 2 percent paraformaldehyde diluted in PBS was added to each well for10 minutes at room temperature and then discarded. The plates were againwashed with PBS containing MgCl₂ and CaCl₂ three times at roomtemperature. Blocking was done with 100 microliters/well of 5 percentmilk in wash buffer (PBS+0.05 percent Tween-20) for 1 hour at roomtemperature. The plates were washed thrice with wash buffer and thehybridoma supernatant was added at 75 microliters/well for 1 hour atroom temperature. The plates were washed 3 times with wash buffer and100 microliters/well of 1/25,000 dilution of goat anti-mouse IgGantibody conjugated to horseradish peroxidase (diluted in PBS containing1 percent milk) was added. After 1 hour incubation at room temperaturethe plates were washed 3 times with wash buffer and 100 microliter/wellof TMB substrate was incubated for 1-3 minutes at room temperature. Thereaction was terminated with 50 microliters/well 2M H₂S0₄ and the plateread at 450 nm with a Perkin-Elmer HTS7000 plate reader. The results astabulated in FIG. 1 were expressed as the number of folds abovebackground compared to an in-house IgG isotype control that haspreviously been shown not to bind to the cell lines tested. Theantibodies from the hybridoma AR52A301.5 showed binding to the ovariancancer cell line OVCAR-3. AR52A301.5 did not display a detectable levelof binding to the normal skin cell line CCD-27sk.

In conjunction with testing for antibody binding, the cytotoxic effectof the hybridoma supernatants was tested in the cell lines: OCC-1,OVCAR-3 and CCD-27sk. Calcein AM was obtained from Molecular Probes(Eugene, Oreg.). The assays were performed according to themanufacturer's instructions with the changes outlined below. Cells wereplated before the assay at the predetermined appropriate density. After2 days, 75 microliters of supernatant from the hybridoma microtitreplates were transferred to the cell plates and incubated in a 5 percentCO₂ incubator for 5 days. The wells that served as the positive controlswere aspirated until empty and 100 microliters of sodium azide (NaN₃) orcycloheximide was added. After 5 days of treatment, the plates were thenemptied by inverting and blotting dry. Room temperature DPBS (Dulbecco'sphosphate buffered saline) containing MgCl₂ and CaCl₂ was dispensed intoeach well from a multichannel squeeze bottle, tapped 3 times, emptied byinversion and then blotted dry. 50 microliters of the fluorescentcalcein dye diluted in DPBS containing MgCl₂ and CaCl₂ was added to eachwell and incubated at 37° C. in a 5 percent CO₂ incubator for 30minutes. The plates were read in a Perkin-Elmer HTS7000 fluorescenceplate reader and the data was analyzed in Microsoft Excel. The resultsare tabulated in FIG. 1. Supernatant from the AR52A301.5 hybridomaproduced specific cytotoxicity of 24 percent on the OVCAR-3 cells. Thiswas 53 percent of the cytotoxicity obtained with the positive controlcycloheximide. Results from FIG. 1 demonstrate that the cytotoxiceffects of AR52A301.5 are proportional to the binding levels on thecancer cell types. There was a greater level of cytotoxicity produced inthe OVCAR-3 cells as compared to the OCC-1 cells, coinciding with thehigher level of binding in the OVCAR-3 cells. As tabulated in FIG. 1,AR52A301.5 did not produce cytotoxicity in the CCD-27sk normal cellline. The known non-specific cytotoxic agents cycloheximide and sodiumazide generally produced cytotoxicity as expected.

EXAMPLE 2 In Vitro Binding

AR52A301.5 monoclonal antibodies were produced by culturing thehybridoma in CL-1000 flasks (BD Biosciences, Oakville, ON) withcollections and reseeding occurring twice/week. Standard antibodypurification procedures with Protein G Sepharose 4 Fast Flow (AmershamBiosciences, Baie d'Urfé, QC) were followed. It is within the scope ofthis invention to utilize monoclonal antibodies that are de-immunized,humanized, chimeric or murine.

Binding of AR52A301.2 to pancreatic (BxPC-3, AsPC-1 and PL45), colon(DLD-1, Lovo, SW1116, HT-29 and Colo-205), breast (MDA-MB-468 andMCF-7), prostate (PC-3 and DU-145), lung (NCI-H520 and A549), esophageal(T.Tn), thyroid (SW579), head and neck (FaDu) and ovarian (OCC-1, C-13,OVCA-429, Sk-OV-3, OV2008, Hey, A2780-cp, A2780-s and OVCAR-3) cancercell lines, and non-cancer cell lines from skin (CCD-27sk) and lung(Hs888.Lu) was assessed by flow cytometry (FACS). All cell lines, exceptfor the majority of ovarian cancer cell lines, were obtained from theAmerican Type Tissue Collection (ATCC, Manassas, Va.). C-13, OV2008,Hey, A2780-cp, A2780-s, OCC-1 and OVCA-429 ovarian cancer cell lineswere obtained from the Ottawa Regional Cancer Center (Ottawa, ON).

Cells were prepared for FACS by initially washing the cell monolayerwith DPBS (without Ca⁺⁺ and Mg⁺⁺). Cell dissociation buffer (INVITROGEN,Burlington, ON) was then used to dislodge the cells from their cellculture plates at 37° C. After centrifugation and collection, the cellswere resuspended in DPBS containing MgCl₂, CaCl₂ and 2 percent fetalbovine serum at 4° C. (staining media) and counted, aliquoted toappropriate cell density, spun down to pellet the cells and resuspendedin staining media at 4° C. in the presence of test antibodies(AR52A301.5) or control antibodies (isotype control, anti-EGFR) at 20micrograms/mL on ice for 30 minutes. Prior to the addition of AlexaFluor 546-conjugated secondary antibody the cells were washed once withstaining media. The Alexa Fluor 546-conjugated antibody in stainingmedia was then added for 30 minutes at 4° C. The cells were then washedfor the final time and resuspended in fixing media (staining mediacontaining 1.5 percent paraformaldehyde). Flow cytometric acquisition ofthe cells was assessed by running samples on a FACSarray™ using theFACSarray™ System Software (BD Biosciences, Oakville, ON). The forward(FSC) and side scatter (SSC) of the cells were set by adjusting thevoltage and amplitude gains on the FSC and SSC detectors. The detectorsfor the fluorescence (Alexa-546) channel was adjusted by runningunstained cells such that cells had a uniform peak with a medianfluorescent intensity of approximately 1-5 units. For each sample,approximately 10,000 gated events (stained fixed cells) were acquiredfor analysis and the results are presented in FIG. 3.

FIG. 2 presents the mean fluorescence intensity fold increase aboveisotype control. Representative histograms of AR52A301.5 antibodies werecompiled for FIG. 3. AR52A301.5 showed strong binding to the pancreaticcancer cell line BxPC-3 (29.4-fold), breast cancer cell line MDA-MB-468(22.7-fold), head and neck cancer cell line FaDu (59.8-fold), ovariancancer cell lines OV2008, OVAR-3 and C-13 (32.7-fold, 22.1-fold and38.7-fold) and the colon cancer cell lines Colo-205 and DLD-1 (38.6-foldand 82.8-fold). Binding was also observed on the ovarian cancer celllines Hey, OCC-1, OVCA-429 and OVCAR-3 (5.9-fold, 17.8-fold, 3.4-foldand 22.1-fold respectively), breast cancer cell line MCF-7 (16.0-fold),prostate cancer cell lines PC-3 and DU-145 (5.0-fold and 3.9-fold),esophageal cancer cell line T.Tn (18.6-fold), colon cancer cell lineHT-29 (6.7-fold) and pancreatic cell line PL45 (12.9-fold) with weakerbinding on the lung cancer cell line NCI-H520 (2.1-fold) and non-cancerlung cell line Hs888.Lu (2.6-fold). Binding to the non-cancer cell linefrom skin (CCD-27sk) was not detectable under these conditions. Thesedata demonstrate that AR52A301.5 exhibited functional specificity inthat although there was clear binding to a variety of cancer cell linesthere was only associated cytotoxicity with some of the lines tested.

EXAMPLE 3 In Vivo Prophylactic Tumor Experiments with BxPC-3 Cells

Examples 1 and 2 demonstrated that AR52A301.5 had anti-cancer propertiesagainst a human cancer cell line with detectable binding across severaldifferent cancer indications. With reference to FIGS. 4 and 5, 6 to 8week old female SCID mice were implanted with 5 million human pancreaticcancer cells (BxPC-3) in 100 microliters saline injected subcutaneouslyin the scruff of the neck. The mice were randomly divided into 3treatment groups of 5. On the day after implantation, 20 mg/kg ofAR52A301.5 test antibody or buffer control was administeredintraperitoneally to each cohort in a volume of 300 microliters afterdilution from the stock concentration with a diluent that contained 2.7mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄. The antibody andcontrol samples were then administered once per week for a period of 7weeks in the same fashion. Tumor growth was measured about every seventhday with calipers for 8 weeks or until individual animals reachedCanadian Council for Animal Care (CCAC) endpoints. Body weights of theanimals were recorded once per week for the duration of the study. Atthe end of the study all animals were euthanised according to CCACguidelines.

AR52A301.5 also prevented tumor growth and reduced tumor burden in an invivo prophylactic model of human pancreatic cancer. On day 55post-implantation, 6 days after the last treatment dose, the mean tumorvolume in the AR52A301.5 treated group was 70 percent less than that ofthe buffer control-treated group (p<0.002; FIG. 4).

There were no clinical signs of toxicity throughout the study. Bodyweight, shown in FIG. 5, was used as a surrogate for well-being andfailure to thrive. Within groups, there was a nonsignificant 10 percentincrease in body weight in the control group over the duration of thestudy. As well, there was a nonsignificant increase in the body weightof the AR52A301.5 treated group; an 8 percent increase from a mean of19.6 g to 21.2 g.

In summary, AR52A301.5 was well-tolerated and decreased the tumor burdenin this human pancreatic cancer xenograft model.

EXAMPLE 4 In Vivo Established Tumor Experiments with BxPC-3 Cells

To further determine the efficacy of AR52A301.5 on the BxPC-3 model ofhuman pancreatic cancer, the antibody was tested on an establishedBxPC-3 xenograft model. With reference to FIGS. 6 and 7, 6 to 8 week oldfemale SCID mice were implanted with 5 million human pancreatic cancercells (BxPC-3) in 100 microliters saline injected subcutaneously in thescruff of the neck. Tumor growth was measured with calipers every week.When the majority of the cohort reached an average tumor volume of 85mm³ (range 56-111) at 33 days post-implantation 9 mice were randomlyassigned into each of 2 treatment groups. AR52A301.5 test antibody orbuffer control was administered intraperitoneally to each cohort, withdosing at 20 mg/kg of antibody in a volume of 300 microliters afterdilution from the stock concentration with a diluent that contained 2.7mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄. The antibodies werethen administered 3 times per week for a total of 10 doses in the samefashion until day 53 post-implantation. Tumor growth was measured aboutevery seventh day with calipers until day 63 post-implantation or untilindividual animals reached the CCAC end-points. Body weights of theanimals were recorded once per week for the duration of the study. Atthe end of the study all animals were euthanised according to CCACguidelines.

AR52A301.5 significantly reduced tumor burden in an established model ofhuman pancreatic cancer. On day 54, AR52A301.5-treated animals had amean tumor volume that was 58 percent of the mean tumor volume incontrol-treated animals (p<0.002; FIG. 6). These results correspond to amean T/C of 51 percent for AR52A301.5

Body weight measured at weekly intervals was used as a surrogate forwell-being and failure to thrive. As seen in FIG. 7, there was nosignificant difference in mean body weight between the twoantibody-treated groups and the control at the end of the study. Inaddition, body weight in all groups did not vary significantly over thecourse of the study.

In summary, AR52A301.5 was well-tolerated and decreased the tumor burdenin this established human pancreatic cancer xenograft model. AR52A301.5has demonstrated efficacy in both a preventative and established modelof human pancreatic cancer.

EXAMPLE 5 In Vivo Prophylactic Tumor Experiments with PL45 Cells

Examples 3 and 4 demonstrated that AR52A301.5 had anti-cancer propertiesagainst a human pancreatic cancer cell line. To determine the efficacyof AR52A301.5 against another human pancreatic cell line, the antibodywas tested on a xenograft model of PL45 human pancreatic cancer. Withreference to FIGS. 8, 9, and 10, 8 to 10 week old female SCID mice wereimplanted with 5 million human pancreatic cancer cells (PL45) in 100microliters PBS solution injected subcutaneously in the scruff of theneck. The mice were randomly divided into 2 treatment groups of 10. Onthe day after implantation, 20 mg/kg of AR52A301.5 test antibody orbuffer control was administered intraperitoneally to each cohort in avolume of 300 microliters after dilution from the stock concentrationwith a diluent that contained 2.7 mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and20 mM Na₂HPO₄. The antibody and control samples were then administeredonce per week for the duration of the study. Tumor growth was measuredabout every 7 day with calipers. The study was completed after 9 dosesof antibody. Body weights of the animals were recorded once per week forthe duration of the study. At the end of the study all animals wereeuthanized according to CCAC guidelines.

AR52A301.5 significantly inhibited tumor growth in the PL45 in vivoprophylactic model of human pancreatic cancer cells. Treatment withARIUS antibody AR52A301.5 reduced the growth of PL45 tumors by 46.8percent (p=0.00068, t-test), compared to the buffer treated group, asdetermined on day 77, 20 days after the last dose of antibody (FIG. 8)when almost all mice in control and antibody-treated group were stillalive. The study was still ongoing at day 102, 45 days after last dose,at which point all mice in the control group had been removed from thestudy due to tumor volume. However, 40 percent of the mice in theAR52A301.5-treated group were still alive at this time-point (FIG. 9).

There were no obvious clinical signs of toxicity throughout the study.Body weight measured at weekly intervals was a surrogate for well-beingand failure to thrive. The mean body weight increased in all groups overthe duration of the study (FIG. 10). The mean weight gain between day 13and day 77 was 3.39 g (17.4 percent) in the control group and 2.86 g(14.5 percent) in the AR52A301.5-treated group. There were nosignificant differences between the groups during the treatment periodor at day 77, 20 days after last the dose.

In summary, AR52A301.5 was well-tolerated and significantly reduced thetumor growth in this human pancreatic cancer xenograft model at day 77.AR52A301.5 treatment also demonstrated increased survival in comparisonto buffer treatment. AR52A301.5 therefore has demonstrated efficacy intwo different models of human pancreatic cancer.

EXAMPLE 6 In Vivo Prophylactic Tumor Experiments with PC-3 Cells

Examples 3, 4 and 5 demonstrated that AR52A301.5 had anti-cancerproperties against two different human pancreatic cancer xenograftmodels. To determine the efficacy of AR52A302.5 against a differenthuman cancer xenograft model, the antibody was tested on a PC-3 prostatecancer xenograft model. With reference to FIGS. 11, 12, and 13, 8 to 10week old female SCID mice were implanted with 5 million human prostatecancer cells (PC-3) in 100 microliters PBS solution injectedsubcutaneously in the scruff of the neck. The mice were randomly dividedinto 2 treatment groups of 10. On the day after implantation, 20 mg/kgof AR52A301.5 test antibody or buffer control was administeredintraperitoneally to each cohort in a volume of 300 microliters afterdilution from the stock concentration with a diluent that contained 2.7mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄. The antibody andcontrol samples were then administered once per week for the duration ofthe study. Tumor growth was measured about every 7 day with calipers.The study was completed after 8 doses of antibody. Body weights of theanimals were recorded once per week for the duration of the study. Atthe end of the study all animals were euthanized according to CCACguidelines when reaching endopoint.

AR52A301.5 inhibited tumor growth in the PC-3 in vivo prophylactic modelof human prostate cancer. Treatment with ARIUS antibody AR52A301.5reduced the growth of PC-3 tumors by 30.9 percent (p=0.01443, t-test),compared to the buffer treated group, as determined on day 32 after 5doses (FIG. 11) when almost all the mice in both the control andantibody-treated group were still alive. The study was still ongoing atday 47, 3 days before the last dose, at which point all mice in thecontrol group had been removed from the study due to tumorvolume/lesions. However, at this time-point 40 percent of the mice inAR52A301.5-treated group were still alive (FIG. 12).

There were no obvious clinical signs of toxicity throughout the study.Body weight measured at weekly intervals was a surrogate for well-beingand failure to thrive. The mean body weight remained relatively constant(FIG. 13). There was no significant difference between the groups duringthe treatment period and or at day 32, after 5 doses.

In summary, AR52A301.5 was well-tolerated and reduced the tumor growthin this human prostate cancer xenograft model. Treatment with antibodyalso demonstrated a survival benefit in comparison to the control group.AR52A301.5 has demonstrated efficacy against two different human cancerindications; pancreatic and prostate.

EXAMPLE 7 In Vivo Prophylactic Tumor Experiments with Colo 205 Cells

Examples 3, 4, 5 and 6 demonstrated that AR52A301.5 had anti-cancerproperties against two different human pancreatic and a prostate cancerxenograft model. To determine the efficacy of AR52A302.5 against adifferent human cancer xenograft model, the antibody was tested on aColo 205 colon cancer xenograft model. With reference to FIGS. 14 and15, 8 to 10 week old female SCID mice were implanted with 5 millionhuman colon cancer cells (Colo 205) in 100 microliters PBS solutioninjected subcutaneously in the right flank of each mouse. The mice wererandomly divided into 2 treatment groups of 10. One day afterimplantation, 20 mg/kg of AR52A301.5 test antibody or buffer control wasadministered intraperitoneally to each cohort in a volume of 300microliters after dilution from the stock concentration with a diluentthat contained 2.7 mM-KCl, 1 mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄.The antibody and control samples were then administered once per weekfor the first two weeks and twice per week for another 3 weeks. Tumorgrowth was measured about every 3-4 days with calipers. The treatmentwas completed after 8 doses of antibody. Body weights of the animalswere recorded when tumors were measured for the duration of the study.At the end of the study all animals were euthanized according to CCACguidelines when reaching endpoint.

AR52A301.5 significantly inhibited tumor growth in the Colo 205 in vivoprophylactic model of human colon cancer. Treatment with ARIUS antibodyAR52A301.5 reduced the growth of Colo 205 colon tumors by 35.9 percent(p=0.01872, t-test), compared to the buffer treated group, as determinedon day 27, 4 days before the last dose of antibody (FIG. 14).

There were no obvious clinical signs of toxicity throughout the study.Body weight measured at weekly intervals was a surrogate for well-beingand failure to thrive. There were no significant differences in meanbody weight between the groups during the treatment period (FIG. 15).

In summary, AR52A301.5 was well-tolerated and significantly inhibitedthe tumor growth in this human colon cancer xenograft model. AR52A301.5has demonstrated efficacy against three different human cancerindications; pancreatic, prostate and colon. Treatment benefits wereobserved in two different well-recognized models of human cancer diseasesuggesting pharmacologic and pharmaceutical benefits of this antibodyfor therapy in other mammals, including man.

EXAMPLE 8 Identification of Cross-Reactivity of Antibody AR52A301.5 withHuman TROP-2

Western blot results suggested that AR52A301.5 cross-reacted with a bandof similar properties to that recognized by the antibody AR47A6.4.2(known to bind to human TROP-2; FIG. 16). To further characterize theantigen recognized by AR52A301.5, this antibody was used as a probe onthe Western blot of an immunocomplex prepared with AR47A6.4.2 from thetotal membrane fraction of MDA-MB-231 (MB-231) cells.

Total cell membranes were prepared from confluent cultures of MB-231breast cancer cells. Media was removed from cell stacks and the cellswere washed with phosphate buffered saline (PBS). Cells were dissociatedwith dissociation buffer (Gibco-BRL; Grand Island, N.Y.) for 20 minutesat 37° C. on a platform shaker. Cells were collected and centrifuged at900 g for 10 minutes at 4° C. After centrifugation, cell pellets werewashed by resuspending in PBS and centrifuging again at 900 g for 10minutes at 4° C. Pellets were then stored at −80° C. until required. Toprepare membranes, cell pellets were thawed and resuspended inhomogenization buffer containing 1 tablet per 50 mL of complete proteaseinhibitor cocktail (Roche; Laval QC) at a ratio of 3 mL buffer per gramof cells. The cell suspension was subjected to homogenization using apolytron homogenizer on ice in order to lyse the cells. The cellhomogenate was centrifuged at 15,000 g for 10 minutes at 4° C. to removethe nuclear particulate. Supernatant was harvested, divided into tubesand then centrifuged at 75,600 g for 90 minutes at 4° C. Supernatant wascarefully removed and each membrane pellet was resuspended inapproximately 5 mL of homogenization buffer. The membrane pellets fromall tubes were combined, divided one more time, and centrifuged at75,600 g for 90 minutes at 4° C. Supernatant was carefully removed andthe pellets were weighed. Solubilization buffer containing 1 percentTriton X-100 was added to the pellets at a ratio of 3 mL buffer per gramof membrane pellet. Membranes were solubilized by shaking on a platformshaker at 300 rpm, for 1 hour on ice. The membrane suspension wascentrifuged at 75,600 g to pellet insoluble material. The supernatant,containing the solubilized membrane proteins, was carefully removed fromthe tubes, assayed for protein concentration, and stored at −80° C.

Total membrane fraction of MB-231 was diluted with 1× RIPA buffer to afinal protein concentration of 1 mg/mL. This sample was divided in twoaliquots of identical size. Each aliquot was incubated withantibody-conjugated Protein G Sepharose, conjugated with eitherAR47A6.4.2 or with the 8A3B.6 isotype control antibody, for 2 hours at4° C. After washing, each antibody-conjugated beads sample was dividedin two identical aliquots. One of the sets of IP samples, and one set ofeach of AR47A6.4.2 and 8A3B.6 conjugated ‘mock IP’ beads wereresuspended with 1× non-reducing SDS-PAGE sample buffer, and a secondset was resuspended in 1× reducing SDS-PAGE sample buffer. After boilingan identical aliquot from each sample was loaded onto replicate lanes of10 percent SDS-polyacrylamide gels. After SDS-PAGE the gels weretransferred onto PVDF membranes and the latter blocked for 1 hour with 5percent milk in TBST followed by incubation with the antibodiesAR47A6.4.2, AR52A301.5, with the isotype control antibody 8A3B.6 or withno primary antibody, in blocking buffer, for 2 hours at roomtemperature. Blots were washed, as described above, and incubated with ahorseradish peroxidase-conjugated goat anti-mouse IgG, Fcfragment-specific, diluted in blocking buffer, and incubated for 1 hourat room temperature. After washing as described above, the blots weredeveloped with the ECL Plus reagent following the manufacturer'srecommendations. The result from this experiment (FIG. 17) demonstratethat the antibody AR52A301.5 cross-reacted specifically with an antigenthat is immunoprecipitated by AR47A6.4.2 and that had the same apparentmolecular weight as the antigen recognized by the latter antibody. Theresults also show that the cross-reactivity of AR47A6.4.2, when used asa probe on a Western blot, is abolished by the boiling of the AR47A6.4.2immunocomplexes with reducing SDS-PAGE sample buffer. However,cross-reactivity of AR52A301.5, with the same immunocomplexes underreducing conditions, was not detectably affected, suggesting that bothantibodies may cross-react with different epitopes on the same antigen.While the epitope recognized by AR47A6.4.2 appears to be adisulfide-bond-dependent conformational epitope, that does not appear tobe the case for the epitope recognized by AR52A301.5.

In order to further establish the nature of the antigen recognized bythe antibody AR52A301.5, Western blots of recombinant human TROP-2 (R&DSystems, Minneapolis, Minn.) prepared under non-reducing conditions forSDS-PAGE, were probed with AR52A301.5 and with AR47A6.4.2. The resultsfrom this experiment show (FIG. 18) that both AR47A6.4.2 and AR52A301.5cross-reacted specifically with the recombinant TROP-2. Therefore we canconclude that the epitope recognized by the monoclonal antibodyAR52A301.5 was also contained within the human TROP-2 antigen.

EXAMPLE 9 Deglycosylation Studies

In order to determine the effects of glycosylation on the binding ofAR52A301.5, deglycosylation reactions were set up as per manufacturer's(Enzymatic Deglycosylation Kit, Prozyme, San Leandro, Calif.)instructions under both denaturing and non-denaturing conditions. Fordenaturing reactions, 0.4 micrograms recombinant TROP-2 (rhTROP-2; R&DSystems, Minneapolis, Minn.) or 100 micrograms of MDA-MB-231 membraneproteins (isolated as described above) were diluted to 30 microliterswith water. 10 microliters of incubation buffer (5×, 0.25 M NaH₂PO₄, pH7.0) and 2.5 microliters of denaturation solution (2 percent SDS, 1 Mbeta-mercaptoethanol) were added, and reactions were boiled for 5minutes. Once reactions cooled to room temperature, 2.5 microliters ofdetergent solution (15 percent NP-40) and 1 microliter of each of thefollowing enzymes were added: N-Glycanase® PNGase F (>5 U/mL), SialidaseA™ (≧5 U/mL), O-Glycanase® (≧1.25 U/mL), beta(1-4) Galactosidase (3U/mL) and beta-N-Acetylglucosaminidase (40 U/mL). Control reactions wereincluded which contained 5 microliters of water instead ofdeglycosylation enzymes. For non-denaturing reactions, 0.4 microgramsrhTROP-2 or 100 micrograms of MDA-MB-231 membrane proteins were dilutedto 35 microliters with water. 10 microliter of incubation buffer wasadded, along with 1 microliter of each enzyme listed above. Controlreactions were included which contained 5 microliters of water insteadof deglycosylation enzymes. All reactions were incubated at 37° C. for24 hours.

Following deglycosylation, reactions were prepared for SDS-PAGE. 16.7microliters of reducing or non-reducing sample loading buffer (4×) wasadded to the denatured and non-denatured reactions, respectively.Samples were boiled for 5 minutes, then cooled to room temperature. 16.7microliters of each reaction was loaded onto quadruplicate 12 percentSDS-PAGE gels. Gels were run at 150 V until the dye front ran off.Proteins were transferred to PVDF membranes overnight at 40 V. Membraneswere blocked with 5 percent milk prepared with TBST (Tris-bufferedsaline with 0.05 percent Tween-20) for 1 hour, followed by incubationwith primary antibodies for 2 hours. Each primary antibody was dilutedto 5 micrograms/mL in 5 percent milk, except anti-human TROP-2, whichwas diluted to 2 micrograms/mL. Blots were incubated with one ofAR52A301.5, anti-human TROP-2 (R&D Systems, Minneapolis, Minn.) or1B7.11 (produced in-house) IgG1 isotype control. Following primaryantibody incubation, blots were washed 3 times, 10 minutes each, withTBST. Blots were incubated with goat anti-mouse IgG Fc HRP secondaryantibody diluted to 1:50,000 in 5 percent milk for 1 hour, then washed 3times, 10 minutes each, with TBST. Blots were developed with ECL PlusWestern Blotting Detection Reagents (GE Healthcare, Life Sciencesformerly Amersham Biosciences; Piscataway, N.J.) and an X-ray developer.

FIGS. 19-21 show the results of the 3 blots probed with AR52A301.5,anti-human TROP-2 and IB7.11 IgG₁ isotype control respectively. FIG. 19(blot probed with AR52A301.5) shows a band of similar size in lane 1.This band is not detectable in FIG. 20 (blot probed with anti-humanTROP-2). The reactive band in the sample from lane 2 (non-denatured anddeglycosylated MB-231 total membrane fraction) shows a shift to around37 kDa and was recognized by AR52A301.5 (FIG. 19) and by the commercialanti-TROP-2 antibody (FIG. 20) blot, but not by the isotype control(FIG. 21). No bands were detected in lanes 5 or 6 (denatured andnon-deglycosylated and deglycosylated MB-231 total membrane fraction) inany of the blots, indicating that the antibodies did not detectably bindto the target protein in the total membrane fraction under reducingconditions.

Recombinant human TROP-2 appears as a very intense, very high molecularweight band (apparent molecular weight larger than 220 kDa) in lanes 3and 4 (non-denatured non-deglycosylated and deglycosylated rhTROP-2,respectively) in FIGS. 19 and 20 (blots probed with AR52A301.5 andanti-human TROP-2, respectively) and correspond to disulfide-bond linkedmultimers of rhTROP-2. Less intense bands appear at ˜70 kDa in lane 3(non-denatured non-deglycosylated rhTROP-2) and ˜55 kDa in lane 4(non-denatured and deglycosylated rhTROP-2) in FIG. 19 (blot probed withAR52A301.5) and correspond to the monomeric forms of rhTROP-2. The shiftin apparent molecular weight of both the multimer and monomer bands fromlarger than 220 kDa and 70 kDa to lower than 220 kDa and 55 kDa,respectively (lanes 3 and 4) result form the loss of carbohydrate groupsdue to deglycosylation. Under reducing conditions (lanes 7 and 8),rhTROP-2 is detected only as the smaller monomeric polypeptide, with adecrease of approximately 20 kDa in apparent molecular weight upontreatment with the glycosidase mixture (FIG. 20). FIG. 21 (blot probedwith the isotype control antibody 1B7.11) does not display reactivity inany of the lanes.

All the anti-human TROP-2 antibodies used, AR52A301.5 and the commercialanti-human TROP-2 antibody, recognized the human TROP-2 antigen in atotal membrane preparation from MDA-MB-231, and the purified recombinanthuman TROP-2, prior to, and after, treatment with a mixture ofglycosidases. This result suggests that the antibodies may recognize anon-carbohydrate epitope, possibly a polypeptide epitope, although it isnot possible to rule out that binding may be occurring to a carbohydrategroup that was not removed by the particular mixture of glycosidasesused in this experiment.

EXAMPLE 10 Competition Experiments

In order to further characterize the binding properties of AR47A6.4.2and AR52A301.5, antibody competition experiments were carried out byWestern blot to determine if AR47A6.4.2 and AR52A301.5 recognize similaror distinct epitopes of TROP-2. Two micrograms of recombinant fusionpolypeptides containing the extracellular domain of human TROP-2 and theFc region of human IgG1, and expressed by the mouse myeloma cell lineNS0 (R&D Systems, Minneapolis, Minn.) were subjected to SDS-PAGE undernon-reducing conditions using preparative well combs that spanned theentire length of each of two 10 percent polyacrylamide gels. Theproteins from the gels were transferred to PVbF membranes at 40V forapproximately 17 hours at 4° C. The membranes were blocked with 5percent skim milk in TBST for one hour at room temperature on a rotatingplatform. The membranes were washed twice with approximately 20 mL ofTBST and were placed in a Western multiscreen apparatus creating twentyseparate channels in which different probing solutions were applied.Previously, biotinylated AR47A6.4.2 and AR52A301.5 had been preparedusing EZ-Link NHS-PEO Solid Phase Biotinylation Kit (Pierce, Rockford,Ill.). Primary antibody solutions were prepared by mixing biotinylatedAR47A6.4.2 or biotinylated AR52A301.5 with varying concentrations ofnon-biotinylated antibodies. Specifically, solutions were preparedcontaining 0.05 micrograms/mL of biotinylated AR52A301.5 in 3 percentskim milk in TBST plus 0.5 micrograms/mL, 5 micrograms/mL, 50micrograms/mL, 500 micrograms/mL or 1000 micrograms/mL ofnon-biotinylated antibody. The non-biotinylated antibodies that wereused were AR52A301.5, AR47A6.4.2 and control antibody 8A3B.6(anti-bluetongue virus; IgG2a, kappa, purified in-house). Solutionscontaining 0.05 micrograms/mL of biotinylated AR47A6.4.2 were preparedwith the same concentrations listed above of the non-biotinylatedantibodies AR52A301.5, AR47A6.4.2 and control antibody 1B7.11 (anti-TNP;IgG1, kappa, 20 μg/mL, purified in-house). A negative control solutionconsisting of three percent skim milk in TBST was added to two channelson each membrane.

The primary antibody solutions were incubated in separate channels onthe membranes for 2 hours at room temperature on a rocking platform.Each channel was washed 3 times with TBST for ten minutes on a rockingplatform. Secondary solution consisting of 0.01 micrograms/mL peroxidaseconjugated streptavidin (Jackson Immunoresearch, West Grove Pa.) in 3percent skim milk in TBST was applied to each channel on the membrane,except for one channel on each membrane to which 3 percent milk in TBSTalone was applied as a negative control. The membranes were incubated insecondary solution for 1 hour at room temperature on a rocking platform.Each channel was washed 3 times with TBST for ten minutes on a rockingplatform. The membranes were removed from the multiscreen apparatus andincubated with an enhanced chemiluminescence detection solution (GEHealthcare, Life Sciences formerly Amersham Biosciences; Piscataway,N.J.) according to manufacturer's directions. The membranes were thenexposed to film and developed.

FIGS. 22 and 23 show the results of the antibody competitionexperiments. Binding of the biotinylated AR52A301.5 was completelyinhibited at a concentration of 50 micrograms/mL and greater ofnon-biotinylated AR52A301.5 (1000× xcess; FIG. 22 lanes 3-7) while thebinding of AR47A6.4.2 was completely inhibited at a concentration of 500micrograms/mL and greater of non-biotinylated AR47A6.4.2 (10000× excess;FIG. 23 lanes 9-13). The binding of biotinylated AR52A301.5 was notinhibited in any of the samples containing IgG2a isotype controlantibody (FIG. 22 lanes 15-19). and the binding of biotinylatedAR47A6.4.2 was not inhibited in any of the samples containing IgG1isotype control antibody (FIG. 23 lanes 15-19). This indicates that theinhibition of binding observed with the biotinylated antibodies mixedwith the same non-biotinylated antibody was due to the occupation ofantigen binding sites by the non-biotinylated antibody, not bynon-specific interactions of excess antibody alone. The binding ofbiotinylated AR52A301.5 was not completely inhibited in any of thesamples containing AR47A6.4.2, and the binding of biotinylatedAR47A6.4.2 was not completely inhibited in any of the samples containingAR52A301.5. In both Western blots however, the reactivity of eachbiotinylated TROP-2 antibody at 5 micrograms/mL, 50 micrograms/mL, 500micrograms/mL, and 1000 micrograms/mL of the other non-biotinylatedTROP-2 antibody was less intense than in the corresponding lanes ofexcess isotype control antibody. These results indicate that the bindingof AR52A301.5 does not prevent the binding of AR47A6.4.2 to TROP-2 andvice versa. Overall, the results of the competition Western blotssuggest that the epitopes of the TROP-2 molecule that are recognized byAR47A6.4.2 and AR52A301.5 are distinct from one and other, although thebinding of one antibody does affect the binding of the other.

EXAMPLE 11 Human Normal Tissue Staining

IHC studies were conducted to characterize the AR52A301.5 antigendistribution in human normal tissues. Slides were postfixed for 10minutes in cold (−20° C.) acetone and then allowed to come to roomtemperature. Slides were rinsed in 4° C. cold phosphate buffered saline(PBS) 3 times for 2 minutes each followed by blocking endogenousperoxidase activity with washing in 3 percent hydrogen peroxide for 10minutes. Slides were then rinsed in PBS 3 times for 5 minutes followedby incubation in Universal blocking solution (Dako, Toronto, Ontario)for 5 minutes at room temperature. AR52A301.5, anti-human muscle actin(Clone HHF35, Dako, Toronto, Ontario), anti-TROP-2 clone 77220.11 (R&DSystem Inc., MN, USA) or isotype control antibody (directed towardsAspergillus niger glucose oxidase, an enzyme which is neither presentnor inducible in mammalian tissues; Dako, Toronto, Ontario) were dilutedin antibody dilution buffer (Dako, Toronto, Ontario) to its workingconcentration (5 micrograms/mL for each antibody except for anti-actinwhich was 0.5 micrograms/mL and commercial TROP-2 was 1 microgram/mL)and incubated overnight for 1 hour at room temperature. The slides werewashed with PBS 3 times for 2 minutes each. Immunoreactivity of theprimary antibodies was detected/visualized with HRP conjugated secondaryantibodies as supplied (Dako Envision System, Toronto, Ontario) for 30minutes at room temperature. Following this step the slides were washedwith PBS 3 times for 5 minutes each and a color reaction developed byadding DAB (3,3′-diaminobenzidine tetrahydrachloride, Dako, Toronto,Ontario) chromogen substrate solution for immunoperoxidase staining for10 minutes at room temperature. Washing the slides in tap waterterminated the chromogenic reaction. Following counterstaining withMeyer's Hematoxylin (Sigma Diagnostics, Oakville, ON), the slides weredehydrated with graded ethanols (75-100 percent) and cleared withxylene. Using mounting media (Dako Faramount, Toronto, Ontario) theslides were coverslipped. Slides were microscopically examined using anAxiovert 200 (Ziess Canada, Toronto, ON) and digital images acquired andstored using Northern Eclipse Imaging Software (Mississauga, ON).Results were read, scored and interpreted by a histopathologist.

Binding of antibodies to 12 human normal organs, ovary, pancreas,thyroid, brain (cerebrum, cerebellum), lung, spleen, uterus, cervix,heart, skin, and skeletal muscle was performed using a human normaltissue screening array (Biochain, CA, USA). The tissue microarraycontained 20 human normal organs; however, 8 of them wereuninterruptible either due to detachment or background staining. FIG. 24presents a summary of the results of AR52A301.5 staining of an array ofhuman normal tissues. AR52A301.5 showed restricted binding to epithelialtissues (endothelium of blood vessels, follicular epithelium of thyroid,acinar and ductal epithelium of pancreas, alveolar epithelium of lung,and epidermal keratinocytes of skin). The antibody also showed equivocalbinding to lymphoid tissue of the spleen and negative binding to neuraltissue of the brain (FIG. 25). Cellular localization was cytoplasmic andmembranous with diffuse staining pattern. AR52A301.5 showed morerestricted binding to human normal tissues than the commercialanti-TROP-2 antibody.

EXAMPLE 12 Multi-Tumor Tissue Staining

IHC studies were conducted to characterize the AR52A301.5 antigenprevalence in human cancers. Slides were transferred from −80 to −20° C.After one hour the slides were postfixed for 10 minutes in cold (−20°C.) acetone and then allowed to come to room temperature. Slides wererinsed in 4° C. cold phosphate buffered saline (PBS) 3 times for 2minutes each followed by blocking endogenous peroxidase activity withwashing in 3 percent hydrogen peroxide for 10 minutes. Slides were thenrinsed in PBS 3 times for 5 minutes followed by incubation in Universalblocking solution (Dako, Toronto, Ontario) for 5 minutes at roomtemperature. AR52A301.10, anti-cytokeratin 7 clone OV-TL 12/30 (Dako,Toronto, Ontario), or isotype control antibody (directed towardsAspergillus niger glucose oxidase, an enzyme which is neither presentnor inducible in mammalian tissues; Dako, Toronto, Ontario) were dilutedin antibody dilution buffer (Dako, Toronto, Ontario) to its workingconcentration 5 micrograms/mL for each antibody except for anti-actinwhich was 0.5 microgram/mL and anti-cytokeratin 7 which was ready to useand incubated for 1 hour at room temperature. The slides were washedwith PBS 3 times for 2 minutes each. Immunoreactivity of the primaryantibodies was detected/visualized with HRP conjugated secondaryantibodies as supplied (Dako Envision System, Toronto, Ontario) for 30minutes at room temperature. Following this step the slides were washedwith PBS 3 times for 5 minutes each and a color reaction developed byadding DAB (3,3′-diaminobenzidine tetrahydrachloride, Dako, Toronto,Ontario) chromogen substrate solution for immunoperoxidase staining for10 minutes at room temperature. Washing the slides in tap waterterminated the chromogenic reaction. Following counterstaining withMeyer's Hematoxylin (Sigma Diagnostics, Oakville, ON), the slides weredehydrated with graded ethanol (75-100 percent) and cleared with xylene.Using mounting media (Dako Faramount, Toronto, Ontario) the slides werecoverslipped. For the pancreatic array (Tri Star, Rockville, Md.) thesame protocol was followed except for the following modifications. Thetissue sections were initially air dried at room temperature for 2 hoursand air dried again for 30 minutes after fixation with acetone. Theendogenous hydrogen peroxide was blocked using 3 percent hydrogenperoxide in methanol for 15 minutes; this step was done after theprimary antibody incubation.

Slides were microscopically examined using an Axiovert 200 (ZiessCanada, Toronto, ON) and digital images acquired and stored usingNorthern Eclipse Imaging Software (Empix, Mississauga, ON). Results wereread, scored and interpreted by a histopathologist.

FIG. 26 presents a summary of the results of AR52A301.5 staining ofvarious human tumors and their corresponding normal tissue sections (8colon cancers, 9 ovarian cancers and 2 normal ovary, 10 breast cancersand 3 normal breast, 9 lung cancers and 2 normal lung, 13 prostatecancers and 3 normal prostate, and 13 pancreatic cancers and 4 normalpancreas). The tissues were distributed on three different tissuemicroarrays (Tri Star, Rockville, Md.). The AR52A301.5 antibody showedmoderate to strong binding to 4/8 (50 percent), 7/9 (78 percent), 7/10(70 percent), 6/9 (67 percent), 12/13 (92 percent) and 2/13 (15 percent)of colon, ovarian, breast, lung, prostate and pancreatic cancers,respectively (FIG. 27). In addition, equivocal to weak binding wasobserved in 3/8 (38 percent), 1/9 (11 percent), 3/10 (30 percent), 3/9(33 percent), and 1/13 (8 percent) colon, ovarian, breast, lung andpancreatic cancer sections, respectively. In all of the tested tumors,the binding was specific for the tumor cells. For the correspondingnormal tissues, the antibody showed no binding to 2/2 ovarian and ⅓breast tissues. Equivocal to weak binding to ½ lung and 4/4 pancreaticnormal tissues and moderate to strong binding to ⅔ breast, ½ lung and3/3 prostate normal tissues was also observed. The binding wasrestricted to epithelial tissues of the normal organs. The positivecontrol antibodies anti-cytokeratin-7 or anti-actin showed the expectedpositive binding to epithelial and muscular tissues, respectively. Thenegative IgG isotype control showed no detectable binding to any of thetested tissues.

EXAMPLE 13 Multi-species Tissue Staining

IHC studies were conducted to characterize the AR52A301.5 antigen crossreactivity in frozen normal tissues of various species in order toselect a preclinical toxicology model. Sections of SCID mouse normaltissues (harvested in house), a rat normal tissue array (Biochain, CA,USA), a multi-species brain array (Biochain, CA, USA) and amulti-species liver array (Biochain, CA, USA) were transferred from −80to −20° C. After one hour the slides were post fixed for 10 minutes incold (−20° C.) acetone and then allowed to come to room temperature.Slides were rinsed in 4° C. cold phosphate buffered saline (PBS) 3 timesfor 2 minutes each followed by blocking endogenous peroxidase activitywith washing in 3 percent hydrogen peroxide for 10 minutes. Slides werethen rinsed in PBS 3 times for 5 minutes followed by incubation inUniversal blocking solution (Dako, Toronto, Ontario) for 5 minutes atroom temperature. AR52A301.5, anti-Grp94 (Stressgen, Victoria, BC,Canada), anti-human muscle actin (Clone HHF35, Dako, Toronto, Ontario)or isotype control antibody (directed towards Aspergillus niger glucoseoxidase, an enzyme which is neither present nor inducible in mammaliantissues; Dako, Toronto, Ontario) was diluted in antibody dilution buffer(Dako, Toronto, Ontario) to its working concentration (5 micrograms/mLfor each antibody except for anti-actin which was 0.5 microgram/mL) andincubated for 1 hour at room temperature. The slides were washed withPBS 3 times for 2 minutes each. Immunoreactivity of the primaryantibodies was detected/visualized with HRP conjugated secondaryantibodies as supplied (Dako Envision System, Toronto, Ontario) for 30minutes at room temperature. Following this step the slides were washedwith PBS 3 times for 5 minutes each and a color reaction developed byadding DAB (3,3′-diaminobenzidine tetrahydrachloride, Dako, Toronto,Ontario) chromogen substrate solution for immunoperoxidase staining for10 minutes at room temperature. Washing the slides in tap waterterminated the chromogenic reaction. Following counterstaining withMeyer's Hematoxylin (Sigma Diagnostics, Oakville, ON), the slides weredehydrated with graded ethanols (75-100 percent) and cleared withxylene. Using mounting media (Dako Faramount, Toronto, Ontario) theslides were coverslipped. For the human, cynomolgus, rabbit, hamster andrhesus individual sections (Biochain, CA, USA) the same protocol wasfollowed with the following modifications. For the first step, thetissue sections were air dried at room temperature for 30 minutes andthen washed with cold PBS without acetone fixation (the sections wereacetone fixed from the manufacturer). The endogenous hydrogen peroxidewas blocked using 3 percent hydrogen peroxide in methanol for 20minutes; this step was done after the primary antibody incubation.

Immunoreactivity of the primary antibodies was detected/visualized withanti-mouse HRP conjugated secondary antibodies as supplied (DakoEnvision System, Toronto, Ontario) for 30 minutes at room temperature.Slides were microscopically examined using an Axiovert 200 (ZiessCanada, Toronto, ON) and digital images acquired and stored usingNorthern Eclipse Imaging Software (Mississauga, ON). Results were read,scored and interpreted by a histopathologist.

Binding of the antibodies to a panel of SCID mouse normal tissues(harvested in house), brain tissues of rat, guinea pig, goat, sheep,chicken, cow, horse, dog and pig (Biochain, CA, USA), liver tissues fromrat, goat, chicken and cow (Biochain, CA, USA) and human, cynomolgus,rhesus, rabbit, hamster and guinea pig individual tissue sections(Biochain, CA, USA) was determined. The positive control antibodyanti-actin (Clone HHF35, Dako, Toronto, Ontario) showed the expectedspecific binding to muscular tissues. The positive control antibodyanti-Grp94 (Stressgen, Victoria, BC) showed the expected positivebinding to predominantly the epithelial tissues. The isotype negativecontrol antibody (Dako, Toronto, Ontario) generally showed no detectablebinding to the tested tissues. Tissue sections that showed obviousbackground staining in the negative control were excluded frominterpretation.

FIG. 28 shows the tabulated results of AR52A301.5 staining of the humanand various species normal tissues. AR52A301.5 showed no binding to thetested rabbit, mouse, rat, guinea pig, goat, sheep, hamster, chicken,cow, horse or pig normal tissues. For the normal dog tissues, there wasdissimilar binding to that observed in the corresponding human tissues.For the cynomolgus normal tissues, AR52A301.5 showed similar tissuespecificity as observed in the corresponding human tissues (FIG. 29) forall of the tested organs except for the ovary in which no binding wasobserved in cynomolgus. For the rhesus normal tissues, AR52A301.5 showedsimilar tissue specificity as observed in the corresponding humantissues for all of the tested organs (FIG. 29). It should be noted thatrhesus normal tissue panel was smaller than what was tested for thecynomolgus. Based on the staining profiles, both the cynomolgus andrhesus monkey are considered to be suitable toxicology models forAR52A301.5.

EXAMPLE 14 AR52A301.5 Murine Sequence 1.0 Cloning Variable Region GenesInto Sequencing Vectors

To facilitate production of antibody chimera, the genes encoding thevariable regions of both heavy and light chains were separately clonedinto the commercial sequencing vector pGEM-T easy (Promega Corp.,Madison, Wis.).

1.1 Isolation of mRNA

Messenger ribonucleic acid (mRNA) was isolated from a culture ofconfluent Master Cell Bank (AR52A301.5) hybridoma cells using Poly ATract System 1000 mRNA extraction kit (Promega Corp., Madison, Wis.).mRNA was stored at −80° C. until required for further use.

1.2 RT-PCR Amplification of Variable Region Genes

Separate reactions were carried out to amplify the light and heavy chainvariable regions. Reverse transcriptase polymerase chain reaction(RT-PCR) synthesized complimentary deoxynucleic acid (cDNA) from themRNA template and then specifically amplified the targeted gene.

For the light chain, 5.0 microliters of mRNA was mixed with 1.0microliter of 20 μmol/microliter MuIgGκV_(L)-3′ primer OL040 and 5.5microliters nuclease free water (Promega Corp., Madison, Wis.). For thelambda light chain, 5.0 microliters of mRNA was mixed with 1.0microliter of 20 μmol/microliter MuIgGλV_(L)-3′ primer OL042 and 5.5microliters nuclease free water (Promega Corp., Madison, Wis.). For thegamma heavy chain, 5 microliters of mRNA was mixed with 1.0 microliterof 20 μmol/microliter MuIgGV_(H)-3′ primer OL023 and 5.5 microlitersnuclease free water (Promega Corp., Madison, Wis.). All three reactionmixes were placed in the pre-heated block of the thermal cycler set at70° C. for 5 minutes. The reaction mixes were then chilled on ice for 5minutes before adding to each 4.0 microliters ImPrommII 5× reactionbuffer (Promega Corp., Madison, Wis.), 0.5 microliters RNasinribonuclease inhibitor (Promega Corp., Madison, Wis.), 2.0 microliters25 mM MgCl₂ (Promega Corp., Madison, Wis.), 1.0 microliter 10 mM dNTPmix (Promega Corp., Madison, Wis.) and 1.0 microliter Improm II reversetranscriptase (Promega Corp., Madison, Wis.). These reaction mixes wereincubated at room temperature for 5 minutes before being transferred toa pre-heated PCR block set at 42° C. for 1 hour. After this time thereverse transcriptase was heat inactivated by incubating at 70° C. in aPCR block for fifteen minutes.

Heavy and light chain sequences were then specifically amplified usingpools of primers (see FIG. 30 for primer sequences; SEQ ID NOS:9-46).The primer working solutions were made up as follows:

-   -   1. 5′ single primer (MuIgV_(H)5′-A and B; MuIgκV_(L)h5′-A, B and        C; MuIgλV_(L)5′-A) contained each primer at a concentration of        20 micromolar;    -   2. 5′ primer pools (MUIgV_(H)5′-C to F; MuIgκV_(L)h5′-D to G)        contained each constituent primer at a concentration of 5        micromolar.

Heavy and light chain sequences were amplified from cDNA. A PCR mastermix was prepared by adding 37.5 microliters 10× Hi-Fi Expand PCR buffer(Roche, Mannheim, Germany), 7.5 microliters 10 mM dNTP mix (Invitrogen,Paisley, UK) and 3.75 microliters Hi-Fi Expand DNA polymerase (Roche,Mannheim, Germany) to 273.75 microliters nuclease free water. Thismaster mix was dispensed in 21.5 microliter aliquots into 15 thin walledPCR reaction tubes, on ice. Into six of these tubes was added 2.5microliters of MuIgV_(H)-3′ reverse transcription reaction mix and 1.0microliters of heavy chain 5′ primer mix A to F. To another seven tubeswas added 2.5 microliters of MuIgκV_(L)-3′ reverse transcriptionreaction and 1.0 microliters of light chain 5′ primer mixes A to G. Intothe final tube was added 2.5 microliter of MuIgλV_(L)-3′ reversetranscription reaction and 1.0 microliters of lambda light chain primerMuIgλV_(L)5′-A. Reactions were placed in the block of the thermal cyclerand heated to 95° C. for 2 minutes. The polymerase chain reaction (PCR)reaction was performed for 40 cycles of 94° C. for 30 seconds, 55° C.for 1 minute and 72° C. for 30 seconds. Finally the PCR products wereheated at 72° C. for 5 minutes, and then stored at 4° C. PCR product waspurified using QIAquick PCR Purification Kit (QIAGEN, Crawley, UK).

FIG. 31 shows the result of the RT-PCR reactions. The heavy chainreactions (lanes 2-7) demonstrate a strong band at 500 bp amplifiedusing MuIgV_(H) 5′-B (lane 3) and MuIgV_(H) 5′-E (lane 6). Light chainreactions (lanes 8-15) demonstrate a strong 450 bp product bandamplified using forward primer MiIgκV_(L) 5′-G (lane 14). PCR productsfrom these reactions were purified using QIAquick PCR Purification Kit(QIAGEN, Crawley, UK).

1.3 Cloning into Sequencing Vectors

Light chain G and heavy chain B and E purified PCR products wereseparately cloned into pGEM-T easy vector using the pGEM-T easy VectorSystem I (Promega Corp., Madison, Wis.). Both the light and heavy chainreactions were prepared by adding 3.0 microliters of purified PCRproduct to 5.0 microliters of 2× ligation buffer, 1.0 microliters pGEM-Teasy vector and 1.0 microliters T4 DNA ligase. Plasmids were transformedinto sub-cloning grade XL1-blue competent E. coli (Stratgene, La Jolla,Calif.) as per the manufacturer's instructions. For all transformations,2.0 microliters of the ligation reaction was used.

100 microliters of transformed cells from each reaction was plated onLuria broth (LB) agar (Q-Biogene, Cambridge, UK) plates containing 50micrograms/mL ampicillin (Sigma, Poole, UK). The plates were invertedand incubated at 37° C. overnight.

Eight clones from each of the three plates were selected and used toinoculate 20 microliters sterile water. A PCR master mix was prepared bymixing 385.2 microliters sterile water, 25.5 microliters DimethylSulphoximide (Sigma, Poole, UK), 51 microliters 10×Taq buffer(Invitrogen, Paisley, UK), 10.2 microliters 10 mM dNTP mix (Invitrogen,Paisley, UK), 5.1 microliters of 50 pmol/microliter primer OL001, 5.1microliters of 50 pmol/microliter primer OL002 and 2.5 microliters TaqDNA polymerase (Invitrogen, Paisley, UK). This master mix was dispensedinto 24 PCR reaction tubes in 19 microliter aliquots. Into each of thesereaction tubes was added 1.0 microliter of the inoculated colonysuspensions. PCR reactions were placed in the block of the thermalcycler and heated to 95° C. for 5 minutes. The polymerase chain reaction(PCR) reaction was performed for 25 cycles of 94° C. for 1 minute, 55°C. for 1 minute and 72° C. for 1 minute. Finally the PCR products wereheated at 72° C. for 10 minutes. 5 microliters from each reaction wasthen run on a 1 percent agarose gel.

FIG. 32 shows the PCR screening reactions from eight colonies ofAR52A301.5V_(H)-B and eight colonies of AR52A301.5V_(H)-E. Seven of theeight V_(H)B colonies, V_(H)B-2 to V_(H)B-8 (lanes 2-8) and one of theeight V_(H)-E colonies, V_(H)E-2 (lane 11) were positive for a 650 bpproduct band indicating the successful cloning of a 500 bp product intopGEM-T easy vector. The remaining V_(H)B and seven V_(H)-E reactionsproduced bands of a different molecular weight indicating a negativeresult for a 500 bp insert.

FIG. 32 also shows the PCR screening reactions from eight colonies ofAR52A301.5V_(L)-G. Four of the eight V_(L)G colonies; V_(L)G-2,V_(L)G-5, V_(L)G-6 and V_(L)G-7 (lanes 19, 22, 23 and 24) were positivefor a 600 bp product band indicating the successful cloning of a 450 bpproduct into pGEM-T easy vector. The remaining 4 V_(L)-G reactionsproduced bands of a different molecular weight indicating a negativeresult for a 450 bp insert.

A maximum of 4 positive colonies from each ligation were chosen toinoculate 5 mL of 2YT (Sigma, Poole, UK) broth containing 50 mg/Lampicillin (Sigma, Poole, UK). Cultures were incubated at 37° C. withshaking overnight. Plasmid DNA was extracted from each culture usingQiagen, QIAprep Spin Miniprep Kit (Qiagen, Crawley, UK).

1.4 DNA Sequencing

Plasmid DNA from nine AR52A301.5V_(L) and V_(H) clones (V_(L) G-2, V_(L)G-5, V_(L) G-6, V_(L) G-7, V_(H) B-2, V_(H) B-3, V_(H) B-4, V_(H) B-5and V_(H) E-2) were sequenced at Geneservice Ltd. DNA sequencingfacility (Cambridge, UK). Sequences are listed in FIGS. 33 and 34 withthe complimentarity determining regions (CDRs) underlined. FIG. 33 showsSEQ ID NO:8 with the underlined CDRs designated SEQ ID NOS:4-6. FIG. 34shows SEQ ID NO:7 with the underlined CDRs designated SEQ ID NOS:1-3.CDR definitions and amino acid sequence numbering is done according toKabat et al. (1991). The Kabat numbering is listed above the amino acidsequence in FIGS. 33 and 34. The hyphen in AR52A301.5 light chain CDR3(FIG. 33) was inserted in order to match the Kabat numbering system.

The correct AR52A301.5V_(L) sequence was found in all 4 of the clonesamplified with 5′ primer MuIgκV_(L)-G. The correct AR52A301.5V_(H)sequence was found in all 4 of the clones amplified with 5′ primerMuIgV_(H)-B. Sequences of products amplified with the 5′ primerMuIgV_(H)-E did not align with murine IgG sequences.

EXAMPLE 15 Isolation of Competitive Binders

Given an antibody, an individual ordinarily skilled in the art cangenerate a competitively inhibiting CDMAB, for example a competingantibody, which is one that recognizes the same epitope (Belanger L etal. Clinica Chimica Acta 48:15-18 (1973)). One method entails immunizingwith an immunogen that expresses the antigen recognized by the antibody.The sample may include but is not limited to tissues, isolatedprotein(s) or cell line(s). Resulting hybridomas could be screened usinga competition assay, which is one that identifies antibodies thatinhibit the binding of the test antibody, such as ELISA, FACS or Westernblotting. Another method could make use of phage display antibodylibraries and panning for antibodies that recognize at least one epitopeof said antigen (Rubinstein J L et al. Anal Biochem 314:294-300 (2003)).In either case, antibodies are selected based on their ability todisplace the binding of the original labeled antibody to at least oneepitope of its target antigen. Such antibodies would therefore possessthe characteristic of recognizing at least one epitope of the antigen asthe original antibody.

EXAMPLE 16 Cloning of the Variable Regions of the AR52A301.5 MonoclonalAntibody

The sequences of the variable regions from the heavy (V_(H)) and light(V_(L)) chains of monoclonal antibody produced by the AR52A301.5hybridoma cell line were determined (Example 14). To generate chimericand humanized IgG, the variable light and variable heavy domains can besubcloned into an appropriate vector for expression.

In another embodiment, AR52A301.5 or its de-immunized, chimeric orhumanized version is produced by expressing a nucleic acid encoding theantibody in a transgenic animal, such that the antibody is expressed andcan be recovered. For example, the antibody can be expressed in a tissuespecific manner that facilitates recovery and purification. In one suchembodiment, an antibody of the invention is expressed in the mammarygland for secretion during lactation. Transgenic animals include but arenot limited to mice, goat and rabbit.

(i) Monoclonal Antibody

DNA encoding the monoclonal antibody (as outlined in Example 1) isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of the monoclonal antibodies).The hybridoma cell serves as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. The DNA also may bemodified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the homologous murinesequences. Chimeric or hybrid antibodies also may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

(ii) Humanized Antibody

A humanized antibody has one or more amino acid residues introduced intoit from a non-human source. These non-human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable domain. Humanization can be performed the method ofWinter and co-workers by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody (Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988); reviewed in Clark,Immunol. Today 21:397-402 (2000)).

A humanized antibody can be prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e. theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

(iii) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. These fragments can be produced by recombinant host cells(reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little etal., Immunol. Today 21:364-370 (2000)). For example, Fab′-SH fragmentscan be directly recovered from E. coli and chemically coupled to formF(ab′)₂ fragments (Carter et al., Biotechnology 10:163-167 (1992)). Inanother embodiment, the F(ab′)₂ is formed using the leucine zipper GCN4to promote assembly of the F(ab′)₂ molecule. According to anotherapproach, Fv, Fab or F(ab′) 2 fragments can be isolated directly fromrecombinant host cell culture.

EXAMPLE 17 A Composition Comprising the Antibody of the PresentInvention

The antibody of the present invention can be used as a composition forpreventing/treating cancer. The composition for preventing/treatingcancer, which comprises the antibody of the present invention, arelow-toxic and can be administered as they are in the form of liquidpreparations, or as pharmaceutical compositions of suitable preparationsto human or mammals (e.g., rats, rabbits, sheep, swine, bovine, feline,canine, simian, etc.) orally or parenterally (e.g., intravascularly,intraperitoneally, subcutaneously, etc.). The antibody of the presentinvention may be administered in itself, or may be administered as anappropriate composition. The composition used for the administration maycontain a pharmacologically acceptable carrier with the antibody of thepresent invention or its salt, a diluent or excipient. Such acomposition is provided in the form of pharmaceutical preparationssuitable for oral or parenteral administration.

Examples of the composition for parenteral administration are injectablepreparations, suppositories, etc. The injectable preparations mayinclude dosage forms such as intravenous, subcutaneous, intracutaneousand intramuscular injections, drip infusions, intraarticular injections,etc. These injectable preparations may be prepared by methods publiclyknown. For example, the injectable preparations may be prepared bydissolving, suspending or emulsifying the antibody of the presentinvention or its salt in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mols) adduct of hydrogenated castor oil)),etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is usually filled in an appropriate ampoule. The suppositoryused for rectal administration may be prepared by blending the antibodyof the present invention or its salt with conventional bases forsuppositories. The composition for oral administration includes solid orliquid preparations, specifically, tablets (including dragees andfilm-coated tablets), pills, granules, powdery preparations, capsules(including soft capsules), syrup, emulsions, suspensions, etc. Such acomposition is manufactured by publicly known methods and may contain avehicle, a diluent or excipient conventionally used in the field ofpharmaceutical preparations. Examples of the vehicle or excipient fortablets are lactose, starch, sucrose, magnesium stearate, etc.

Advantageously, the compositions for oral or parenteral use describedabove are prepared into pharmaceutical preparations with a unit dosesuited to fit a dose of the active ingredients. Such unit dosepreparations include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid compoundcontained is generally 5 to 500 mg per dosage unit form; it is preferredthat the antibody described above is contained in about 5 to about 100mg especially in the form of injection, and in 10 to 250 mg for theother forms.

The dose of the aforesaid prophylactic/therapeutic agent or regulatorcomprising the antibody of the present invention may vary depending uponsubject to be administered, target disease, conditions, route ofadministration, etc. For example, when used for the purpose oftreating/preventing, e.g., breast cancer in an adult, it is advantageousto administer the antibody of the present invention intravenously in adose of about 0.01 to about 20 mg/kg body weight, preferably about 0.1to about 10 mg/kg body weight and more preferably about 0.1 to about 5mg/kg body weight, about 1 to 5 times/day, preferably about 1 to 3times/day. In other parenteral and oral administration, the agent can beadministered in a dose corresponding to the dose given above. When thecondition is especially severe, the dose may be increased according tothe condition.

The antibody of the present invention may be administered as it standsor in the form of an appropriate composition. The composition used forthe administration may contain a pharmacologically acceptable carrierwith the aforesaid antibody or its salts, a diluent or excipient. Such acomposition is provided in the form of pharmaceutical preparationssuitable for oral or parenteral administration (e.g., intravascularinjection, subcutaneous injection, etc.). Each composition describedabove may further contain other active ingredients. Furthermore, theantibody of the present invention may be used in combination with otherdrugs, for example, alkylating agents (e.g., cyclophosphamide,ifosfamide, etc.), metabolic antagonists (e.g., methotrexate,5-fluorouracil, etc.), anti-tumor antibiotics (e.g., mitomycin,adriamycin, etc.), plant-derived anti-tumor agents (e.g., vincristine,vindesine, Taxol®, etc.), cisplatin, carboplatin, etoposide, irinotecan,etc. The antibody of the present invention and the drugs described abovemay be administered simultaneously or at staggered times to the patient.

The method of treatment described herein, particularly for cancers, mayalso be carried out with administration of other antibodies orchemotherapeutic agents. For example, an antibody against EGFR, such asERBITUX® (cetuximab), may also be administered, particularly whentreating colon cancer. ERBITUX® has also been shown to be effective fortreatment of psoriasis. Other antibodies for combination use includeHerceptin® (trastuzumab) particularly when treating breast cancer,AVASTIN® particularly when treating colon cancer and SGN-15 particularlywhen treating non-small cell lung cancer. The administration of theantibody of the present invention with other antibodies/chemotherapeuticagents may occur simultaneously, or separately, via the same ordifferent route.

The chemotherapeutic agent/other antibody regimens utilized include anyregimen believed to be optimally suitable for the treatment of thepatient's condition. Different malignancies can require use of specificanti-tumor antibodies and specific chemotherapeutic agents, which willbe determined on a patient to patient basis. In a preferred embodimentof the invention, chemotherapy is administered concurrently with or,more preferably, subsequent to antibody therapy. It should beemphasized, however, that the present invention is not limited to anyparticular method or route of administration.

The preponderance of evidence shows that AR52A301.5 mediate anti-cancereffects and prolongs survival through ligation of epitopes present onTROP-2. It has been shown, in Examples 8 and 9 that AR52A301.5antibodies can be used to immunoprecipitate the cognate antigen fromexpressing cells such as MDA-MB-231 cells. Further it has been shown, inExamples 1, 2 and 11-13 that the AR52A301.5 antibody could be used indetection of cells and/or tissues which express a TROP-2 antigenicmoiety which specifically binds thereto, utilizing techniquesillustrated by, but not limited to FACS, cell ELISA or IHC.

Thus, it could be shown that the immunoprecipitated AR52A301.5 antigencan inhibit the binding of AR52A301.5 to such cells or tissues usingFACS, cell ELISA or IHC assays. Further, as with the AR52A301.5antibody, other anti-TROP-2 antibodies could be used toimmunoprecipitate and isolate other forms of the TROP-2 antigen, and theantigen can also be used to inhibit the binding of those antibodies tothe cells or tissues that express the antigen using the same types ofassays.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementof parts herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specification.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Anyoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A humanized antibody of the isolated monoclonal antibody produced bythe hybridoma deposited with the IDAC as accession number 141205-03 oran antigen binding fragment produced from said humanized antibody.
 2. Achimeric antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the IDAC as accession number 141205-03 or anantigen binding fragment produced from said chimeric antibody.
 3. Amethod for initiating antibody induced cytotoxicity of cancerous cellsin a tissue sample selected from a human pancreatic, ovarian, prostateor colon tumor comprising: providing a tissue sample from said humanpancreatic, ovarian, prostate or colon tumor providing the isolatedmonoclonal antibody produced by the hybridoma deposited with the IDAC asaccession number 141205-03, the humanized antibody of the isolatedmonoclonal antibody produced by the hybridoma deposited with the IDAC asaccession number 141205-03, the chimeric antibody of the isolatedmonoclonal antibody produced by the hybridoma deposited with the IDAC asaccession number 141205-03, or an antigen binding fragment of any of theforegoing, characterized by an ability to competitively inhibit bindingof said isolated monoclonal antibody to its target antigen; andcontacting said isolated monoclonal antibody, said humanized antibody,said chimeric antibody or said antigen binding fragment thereof withsaid tissue sample; wherein binding of said isolated monoclonalantibody, said humanized antibody, said chimeric antibody or saidantigen binding fragment thereof with said tissue sample inducescytotoxicity.
 4. An antigen binding fragment of the humanized antibodyof claim
 1. 5. An antigen binding fragment of the chimeric antibody ofclaim
 2. 6. A method of reduction of a human pancreatic, ovarian,prostate or colon tumor susceptible to antibody induced cytotoxicity ina mammal, wherein said human pancreatic, ovarian, prostate or colontumor expresses at least one epitope of an antigen which specificallybinds to the isolated monoclonal antibody produced by the hybridomadeposited with the IDAC as accession number 141205-03 or an antigenbinding fragment thereof, which antigen binding fragment ischaracterized by an ability to competitively inhibit binding of saidisolated monoclonal antibody to its target antigen, comprisingadministering to said mammal said monoclonal antibody or said antigenbinding fragment thereof in an amount effective to result in a reductionof said mammal's pancreatic, ovarian, prostate or colon tumor burden. 7.The method of claim 6 wherein said isolated monoclonal antibody isconjugated to a cytotoxic moiety.
 8. The method of claim 7 wherein saidcytotoxic moiety is a radioactive isotope.
 9. The method of claim 6wherein said isolated monoclonal antibody or antigen binding fragmentthereof activates complement.
 10. The method of claim 6 wherein saidisolated monoclonal antibody or antigen binding fragment thereofmediates antibody dependent cellular cytotoxicity.
 11. The method ofclaim 6 wherein said isolated monoclonal antibody is a humanizedantibody of the isolated monoclonal antibody produced by the hybridomadeposited with the IDAC as accession number 141205-03.
 12. The method ofclaim 6 wherein said isolated monoclonal antibody is a chimeric antibodyof the isolated monoclonal antibody produced by the hybridoma depositedwith the IDAC as accession number 141205-03.
 13. A monoclonal antibodywhich specifically binds to the same epitope or epitopes as the isolatedmonoclonal antibody produced by the hybridoma deposited with the IDAC asaccession number 141205-03.
 14. A method of reduction of a humanpancreatic, ovarian, prostate or colon tumor in a mammal, wherein saidhuman tumor expresses at least one epitope of an antigen whichspecifically binds to the isolated monoclonal antibody produced by thehybridoma deposited with the IDAC as accession number 141205-03 or anantigen binding fragment thereof, which antigen binding fragment ischaracterized by an ability to competitively inhibit binding of saidisolated monoclonal antibody to its target antigen, comprisingadministering to said mammal said monoclonal antibody or antigen bindingfragment thereof in an amount effective to result in a reduction of saidmammal's pancreatic, ovarian, prostate or colon tumor burden.
 15. Themethod of claim 14 wherein said isolated monoclonal antibody isconjugated to a cytotoxic moiety.
 16. The method of claim 15 whereinsaid cytotoxic moiety is a radioactive isotope.
 17. The method of claim14 wherein said isolated monoclonal antibody or antigen binding fragmentthereof activates complement.
 18. The method of claim 14 wherein saidisolated monoclonal antibody or antigen binding fragment thereofmediates antibody dependent cellular cytotoxicity.
 19. The method ofclaim 14 wherein said isolated monoclonal antibody is a humanizedantibody of the isolated monoclonal antibody produced by the hybridomadeposited with the IDAC as accession number 141205-03.
 20. The method ofclaim 14 wherein said isolated monoclonal antibody is a chimericantibody of the isolated monoclonal antibody produced by the hybridomadeposited with the IDAC as accession number 141205-03.
 21. A method ofreduction of a human pancreatic, ovarian, prostate or colon tumor in amammal, wherein said human pancreatic, ovarian, prostate or colon tumorexpresses at least one epitope of an antigen which specifically binds tothe isolated monoclonal antibody produced by the hybridoma depositedwith the IDAC as accession number 141205-03 or an antigen bindingfragment thereof, which antigen binding fragment is characterized by anability to competitively inhibit binding of said isolated monoclonalantibody to its target antigen, comprising administering to said mammalsaid monoclonal antibody or antigen binding fragment thereof inconjunction with at least one chemotherapeutic agent in an amounteffective to result in a reduction of said mammal's pancreatic, ovarian,prostate or colon tumor burden.
 22. The method of claim 21 wherein saidisolated monoclonal antibody is conjugated to a cytotoxic moiety. 23.The method of claim 22 wherein said cytotoxic moiety is a radioactiveisotope.
 24. The method of claim 21 wherein said isolated monoclonalantibody or antigen binding fragment thereof activates complement. 25.The method of claim 21 wherein said isolated monoclonal antibody orantigen binding fragment thereof mediates antibody dependent cellularcytotoxicity.
 26. The method of claim 21 wherein said isolatedmonoclonal antibody is a humanized antibody of the isolated monoclonalantibody produced by the hybridoma deposited with the IDAC as accessionnumber 141205-03.
 27. The method of claim 21 wherein said isolatedmonoclonal antibody is a chimeric antibody of the isolated monoclonalantibody produced by the hybridoma deposited with the IDAC as accessionnumber 141205-03.
 28. A binding assay to determine a presence ofcancerous cells in a tissue sample selected from a human tumor, which isspecifically bound by the isolated monoclonal antibody produced byhybridoma cell line AR52A301.5 having IDAC Accession No. 141205-03, thehumanized antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the IDAC as accession number 141205-03 or thechimeric antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the IDAC as accession number 141205-03,comprising: providing a tissue sample from said human tumor; providingat least one of said isolated monoclonal antibody, said humanizedantibody, said chimeric antibody or antigen binding fragment thereofthat recognizes the same epitope or epitopes as those recognized by theisolated monoclonal antibody produced by a hybridoma cell lineAR52A301.5 having IDAC Accession No. 141205-03; contacting at least onesaid provided antibodies or antigen binding fragment thereof with saidtissue sample; and determining binding of said at least one providedantibody or antigen binding fragment thereof with said tissue sample;whereby the presence of said cancerous cells in said tissue sample isindicated.
 29. Use of monoclonal antibodies for reduction of humanpancreatic, ovarian, prostate or colon tumor burden, wherein said humanpancreatic, ovarian, prostate or colon tumor expresses at least oneepitope of an antigen which specifically binds to the isolatedmonoclonal antibody produced by the hybridoma deposited with the IDAC asaccession number 141205-03 or an antigen binding fragment thereof, whichantigen binding fragment is characterized by an ability to competitivelyinhibit binding of said isolated monoclonal antibody to its targetantigen, comprising administering to said mammal said monoclonalantibody or antigen binding fragment thereof in an amount effective toresult in a reduction of said mammal's human pancreatic, ovarian,prostate or colon tumor burden.
 30. The method of claim 29 wherein saidisolated monoclonal antibody is conjugated to a cytotoxic moiety. 31.The method of claim 30 wherein said cytotoxic moiety is a radioactiveisotope.
 32. The method of claim 29 wherein said isolated monoclonalantibody or antigen binding fragment thereof activates complement. 33.The method of claim 29 wherein said isolated monoclonal antibody orantigen binding fragment thereof mediates antibody dependent cellularcytotoxicity.
 34. The method of claim 29 wherein said isolatedmonoclonal antibody is a humanized antibody of the isolated monoclonalantibody produced by the hybridoma deposited with the IDAC as accessionnumber 141205-03.
 35. The method of claim 29 wherein said isolatedmonoclonal antibody is a chimeric antibody of the isolated monoclonalantibody produced by the hybridoma deposited with the IDAC as accessionnumber 141205-03.
 36. Use of monoclonal antibodies for reduction ofhuman pancreatic, ovarian, prostate or colon tumor burden, wherein saidhuman pancreatic, ovarian, prostate or colon tumor expresses at leastone epitope of an antigen which specifically binds to the isolatedmonoclonal antibody produced by the hybridoma deposited with the IDAC asaccession number 141205-03 or an antigen binding fragment thereof, whichantigen binding fragment is characterized by an ability to competitivelyinhibit binding of said isolated monoclonal antibody to its targetantigen, comprising administering to said mammal said monoclonalantibody or antigen binding fragment thereof, in conjunction with atleast one chemotherapeutic agent in an amount effective to result in areduction of said mammal's human pancreatic, ovarian, prostate or colontumor burden.
 37. The method of claim 36 wherein said isolatedmonoclonal antibody is conjugated to a cytotoxic moiety.
 38. The methodof claim 37 wherein said cytotoxic moiety is a radioactive isotope. 39.The method of claim 36 wherein said isolated monoclonal antibody orantigen binding fragment thereof activates complement.
 40. The method ofclaim 36 wherein said isolated monoclonal antibody or antigen bindingfragment thereof mediates antibody dependent cellular cytotoxicity. 41.The method of claim 36 wherein said isolated monoclonal antibody is ahumanized antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the IDAC as accession number 141205-03.
 42. Themethod of claim 36 wherein said isolated monoclonal antibody is achimeric antibody of the isolated monoclonal antibody produced by thehybridoma deposited with the IDAC as accession number 141205-03.
 43. Acomposition effective for treating a human pancreatic, ovarian, prostateor colon tumor comprising in combination: an antibody or antigen bindingfragment of any one of claims 1, 2, 4, 5, 13, 45 or 46; a conjugate ofsaid antibody or an antigen binding fragment thereof with a memberselected from the group consisting of cytotoxic moieties, enzymes,radioactive compounds, cytokines, interferons, target or reportermoieties and hematogenous cells; and a requisite amount of apharmaceutically acceptable carrier; wherein said composition iseffective for treating said human pancreatic, ovarian, prostate or colontumor.
 44. An assay kit for detecting the presence of a human canceroustumor, wherein said human cancerous tumor expresses at least one epitopeof an antigen which specifically binds to the isolated monoclonalantibody produced by the hybridoma deposited with the IDAC as accessionnumber 141205-03 or an antigen binding fragment thereof, which antigenbinding fragment is characterized by an ability to competitively inhibitbinding of said isolated monoclonal antibody to its target antigen, thekit comprising the isolated monoclonal antibody produced by thehybridoma deposited with the IDAC as accession number 141205-03 or anantigen binding fragment thereof, and means for detecting whether themonoclonal antibody, or an antigen binding fragment thereof, is bound toa polypeptide whose presence, at a particular cut-off level, isdiagnostic of said presence of said human cancerous tumor.
 45. Anisolated monoclonal antibody or antigen binding fragment thereof, whichspecifically binds to human TROP-2, in which the isolated monoclonalantibody or antigen binding fragment thereof reacts with the sameepitope or epitopes of human TROP-2 as the isolated monoclonal antibodyproduced by a hybridoma cell line AR52A301.5 having IDAC Accession No.141205-03; said isolated monoclonal antibody or antigen binding fragmentthereof being characterized by an ability to competitively inhibitbinding of said isolated monoclonal antibody to its target human TROP-2antigen.
 46. An isolated monoclonal antibody or antigen binding fragmentthereof that recognizes the same epitope or epitopes as those recognizedby the isolated monoclonal antibody produced by the hybridoma cell lineAR52A301.5 having IDAC Accession No 141205-03; said monoclonal antibodyor antigen binding fragment thereof being characterized by an ability tocompetitively inhibit binding of said isolated monoclonal antibody toits target epitope or epitopes.
 47. A process for reduction of a humanpancreatic, ovarian, prostate or colon tumor which expresses at leastone epitope of human TROP-2 antigen which is specifically bound by theisolated monoclonal antibody produced by hybridoma cell line AR52A301.5having IDAC Accession No. 141205-03, comprising: administering to anindividual suffering from said human tumor, at least one isolatedmonoclonal antibody or antigen binding fragment thereof that binds thesame epitope or epitopes as those bound by the isolated monoclonalantibody produced by the hybridoma cell line AR52A301.5 having IDACAccession No. 141205-03; wherein binding of said epitope or epitopesresults in a reduction of pancreatic, ovarian, prostate or colon tumorburden.
 48. A process for reduction of a human pancreatic, ovarian,prostate or colon tumor which expresses at least one epitope of humanTROP-2 antigen which is specifically bound by the isolated monoclonalantibody produced by hybridoma cell line AR52A301.5 having IDACAccession No. 141205-03, comprising: administering to an individualsuffering from said human tumor, at least one isolated monoclonalantibody or antigen binding fragment thereof, that binds the sameepitope or epitopes as those bound by the isolated monoclonal antibodyproduced by the hybridoma cell line AR52A301.5 having IDAC Accession No.141205-03; in conjunction with at least one chemotherapeutic agent;wherein said administration results in a reduction of tumor burden. 49.A binding assay to determine the presence of cells which express TROP-2which is specifically recognized by the isolated monoclonal antibodyproduced by the hybridoma cell line AR52A301.5 having IDAC Accession No.141205-03, or an antigen binding fragment produced from said isolatedmonoclonal antibody comprising: providing a cell sample; providing theisolated monoclonal antibody produced by the hybridoma cell lineAR52A301.5 having IDAC Accession No. 141205-03 or said antigen bindingfragment produced from the isolated monoclonal antibody; contacting saidisolated monoclonal antibody or said antigen binding fragment with saidcell sample; and determining binding of said isolated monoclonalantibody or antigen binding fragment thereof with said cell sample;whereby the presence of cells which express an antigen of TROP-2 whichis specifically bound by said isolated monoclonal antibody or saidantigen binding fragment is determined.
 50. A method of extendingsurvival and delaying disease progression by treating a humanpancreatic, ovarian, prostate or colon tumor in a mammal, wherein saidtumor expresses an antigen which specifically binds to the isolatedmonoclonal antibody produced by the hybridoma cell line AR52A301.5having IDAC Accession No. 141205-03, or an antigen binding fragmentproduced from said isolated monoclonal antibody comprising administeringto said mammal said monoclonal antibody in an amount effective to reducesaid mammal's tumor burden, whereby disease progression is delayed andsurvival is extended.
 51. A method of extending survival and delayingdisease progression by treating a human pancreatic, ovarian, prostate orcolon tumor in a mammal, wherein said tumor expresses TROP-2 whichspecifically binds to the isolated monoclonal antibody produced by thehybridoma cell line AR52A301.5 having IDAC Accession No. 141205-03, or aTROP-2 binding fragment produced from said isolated monoclonal antibodycomprising administering to said mammal said monoclonal antibody in anamount effective to reduce said mammal's tumor burden, whereby diseaseprogression is delayed and survival is extended.
 52. A compositioneffective for treating a human pancreatic, ovarian, prostate or colontumor comprising in combination: an antibody or antigen binding fragmentof any one of claims 1, 2, 4, 5, 13, 45 or 46; and a requisite amount ofa pharmaceutically acceptable carrier; wherein said composition iseffective for treating said human pancreatic, ovarian, prostate or colontumor.
 53. A composition effective for treating a human pancreatic,ovarian, prostate or colon tumor comprising in combination: a conjugateof an antibody or antigen binding fragment of any one of claims 1, 2, 4,5, 13, 45 or 46 with a member selected from the group consisting ofcytotoxic moieties, enzymes, radioactive compounds, cytokines,interferons, target or reporter moieties and hematogenous cells; and arequisite amount of a pharmaceutically acceptable carrier; wherein saidcomposition is effective for treating said human pancreatic, ovarian,prostate or colon tumor.